Inhibitors of multidrug transporters

ABSTRACT

The present invention relates generally to the fields of bacteriology and mycology. More particularly, the present invention provides novel inhibitors of multidrug transport proteins that may be used in combination with existing antibacterial agent and/or antifungal agents to increase the toxic effects of the antimicrobial agents. More specifically the present invention provides methods and compositions for enhancing the antibacterial action of fluoroquinolones by administering fluoroquinolones in combination with an inhibitor of multidrug transporters and of enhancing the antifungal action of azole antifungal agents by administering an azole antifungal agent in combination with an inhibitor of multidrug transporters. Compositions comprising indole, urea, quinoline or aromatic amide based inhibitors also are disclosed.

The present application claims priority to U.S. Provisional PatentApplication Serial No. 60/110,841, filed Dec. 4, 1998. The entire textof the above-referenced disclosure is specifically incorporated byreference herein without disclaimer. The government may own rights inthe present invention pursuant to grant number GM55449-01 and1R43AI43076-01 and GM55449-02 from the National Institutes of Health.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of bacteriologyand mycology. More particularly, the present invention provides methodsand compositions for increasing the effectiveness of existingantibiotics and antifungal agents and methods of overcoming bacterialand fungal resistance.

2. Description of Related Art

Gram positive organisms, particularly Staphylococci, Streptococci, andEnterococci, are increasingly seen as the major aetiological agents ininfectious diseases. In the hospital setting, Staphylococcus aureus andEnterococcus faecalis account for more than 50% of isolates from bloodstream infections (Cormican and Jones, 1996). In community-acquiredinfections Streptococcus pneumoniae remains a leading cause of illnessand death (Centers for Disease Control, 199). The ongoing and rapidemergence and spread of antibiotic resistance in these organism is thusa problem of crisis proportions.

One of the major impediments in treating Gram-positive infections istheir limited susceptibility to fluoroquinolones, the latest addition tothe arsenal of antibiotics. Since their introduction in the mid-1980s,fluoroquinolone antibiotics, have become the most used class ofantibiotics in the world (Acar and Goldstein, 1997). One suchantibiotic., ciprofloxacin (Davis et al., 1996), accounts for 90% of allquinolones used in medicine, Because of its spectrum of activity, oralavailability, and relatively low cost. ciprofloxacin has been used fortreating a wide range of infections, including those of unknownetiology. In 1996, three new indications for the use of ciprofloxacinwere approved suggesting that the use of this antibiotic will continuefor many years to come.

Although being highly active against most Gram negative microorganisms(MIC₉₀ in the range of 0.1 μg/ml), ciprofloxacin is less effectiveagainst Gram positive infections, particularly aerobic Gram positivecocci. The MIC₉₀ values for S. aureus, E. faecalis and S. pneumoniae arein the range of 1-5 μg/ml, whereas the achievable tissue concentrationof ciprofloxacin is only 4 μg/ml (Davis et al., 1996). The highintrinsic resistance to ciprofloxacin, and the extensive use ofquinolones both in human and veterinary medicine, has led to theemergence and dissemination of ciprofloxacin-resistant Gram-positivestrains. This limitation has led to the quest for new, more effectivefluoroquinolones.

Antibiotic resistance is mediated, at least in part, by the efflux ofdrugs from target cells by multidruy transporters (MDTs). Thesetransporters promote the active efflux of a wide variety of drugs,including fluoroquinolone antibiotics, from the bacterial cells that areresponsible for the particular infection. In 1991, Neyfakh et al.published the first description of a chromosomally-encoded bacterialmultidrug transporter, Bmr, of the Gram positive bacteria Bacillussubtilis. Since then, practically every bacterial species analyzed,including pathogenic species such as Escherichia, Pseudomonas,Mycobacteria, etc. (Lomovskaya and Lewis, 1992; Poole et al., 1993;Takiff et al., 1996, reviewed in Nikaido, 1994; Lewis, 1994), has beenshown to express one, or even several multidrug transporters. Forexample, B subtilis expresses at least three multidrug transporters,homologous Bmr and Blt (Ahmed et al., 1995) and an evolutionarily moredistant Bmr3 (Ohki and Murata, 1997). Bmr and its close homolog inStaphylococcus aureus, NorA, promote the efflux of a variety ofbacteriotoxic compounds, including ethidium bromide, rhodamine,acridines, tetraphenylphosphonium and puromycin, with fluoroquinoloneantibiotics being one of the best transporter substrates (Yoshida etal., 1990; Neyfakh, 1992; Neyfakh et al., 1993). Importantly, drugefflux mediated by the Bmr and NorA transporters can be completelyinhibited by the plant alkaloid reserpine, which by itself is not toxicto bacteria (Neyfakh et al.; Neyfakh, 1993).

Multidrug transporters also play an important role in both the intrinsicand acquired resistance of important fungal pathogens to antifuingalagents. Particularly, multidrug transporters contribute to theresistance of Candida albicans, the fourth leading cause of allhospital-acquired infections, to azole antifungal agents.

There is little knowledge regarding the physiological role of multidrugtransporters or the mechanism of their action; nevertheless thesetransporters appear to play an important role in the intrinsicresistance of bacterial cells to toxins and antibiotics. Inactivation ofthe chromosomal transporter genes usually leads to a dramatic increasein the sensitivity of bacteria to the transporter substrates (Poole etal., 1993; Ahmed et al., 1994; Okusu et al., 1996; Yamada et al., 1997).Disruption of the Bmr gene in B. subtilis, or the inhibition of the Bmrtransporter with reserpine, reduces the minimal inhibitory concentration(MIC) of norfloxacin, a typical fluoroquinolone antibiotic, by a factorof five (Neyfakh, 1992). Similarly, multidrug transporters contributesignificantly to the intrinsic fluoroquinolone resistance of Grampositive pathogenic cocci. Yamada et al. (1997) have recently shown thatgenetic disruption of the NorA gene increases the susceptibility of S.aureus to norfloxacin and ciprofloxacin by eight and four fold,respectively. Reserpine, which inhibits NorA-mediated drug efflux,reduces the MIC of norfloxacin for wild-type S. aureus by at leastfour-fold (Markham and Neyfakh, 1996; Kaatz and Seo, 1995). Although themultidrug transporter of S. pneumoniae has not yet been identified, itsexistence is strongly supported by physiological data (Baranova andNeyfakh, 1997; Zeller et al., 1997; Brenwald et al.; 1997). Furthermore,reserpine has been shown to reduce the MIC of norfloxacin andciprofloxacin for wild-type S. pneumonae by the factor of 2-3 (Baranovaand Neyfakh, 1997). In E. faecalis the active efflux of fluoroquinoloneshas been demonstrated biochemically (Lynch et al., 1997) and, again,reserpine provides a two-fold increase in their susceptibility tofluoroquinolones.

In addition to being involved in the intrinsic resistance ofGram-positive cocci to fluoroquinolones, multidrug transporterscontribute to the acquired resistance, which is selected upon exposureto these antibiotics. In S. aureus and S. pneumoniae, the acquiredresistance has so far been attributed mainly to the sequentialacquisition of mutations in the targets of fluoroquinolone action,topoisomerase IV and DNA gyrase (Cambau and Gutman, 1993; Ferrero etal., 1994; Munoz and De La Campa, 1996; Tankovi, 1996). From the limitedstudies of fluoroquinolone resistance mechanisms in E. faecalis, itappears that mutations of gyrase are present in at least some high levetresistant isolates Korten et al., 1994). However, it has become apparentin recent years that these mechanisms of acquired resistance arecomplemented by over-cxpression of multidrug transporters. Suchoverexpression can result from either amplification of the transportergene (Neyfakh, 1991); or mutations in the regulatory regions of thesegenes or regulatory proteins controlling their transcription (Ahmed etal., 1995; Kaatz and Seo, 1995).

Overexpression of the NorA multidrug transporter has been reported forstrains of S. aureus selected for fluoroquinolone resistance both invitro (Yoshida et al., 1990; Kaatz et al., 1990) and in vivo (Trucksiset al., 1991). From the discussion above it is clear that multidrugtransporters present a major impediment to the treatment of Grampositive pathogenic insult. There exists a need for drug(s) that maycircumvent these transporters to be useful in treatment regimens.

SUMMARY OF THE INVENTION

In order to meet the objectives of the present invention, there areprovided methods of enhancing the antimicrobial action of antimicrobialagents by inhibiting the multidrug transporters in the microbes. Aspecific embodiment of the present invention contemplates a method forenhancing the antibacterial action of fluoroquinolones comprisingcontacting a bacterium with an inhibitor of NorA, wherein said inhibitoris an indole, a urea, an aromatic amide or a quinoline.

In more particular embodiments, the inhibitor is an indole that has thegeneral formula:

wherein R₁ is phenyl, 2-naphthyl, o-anisole, R₂ is H or CH₃, R₁ and R₂are two naphthyl groups fused to the indole ring, R₃ is H, R₄ is NO₂,SO₃H, NH₂ and CF₃ or CCl₃, R₅ is H, and R₆ is H. More particularly, theR₁ may be a phenyl group and R₄ may be SO₃H or NO₂. In otherspecifically preferred embodiments, the R₁ may be 2-naphthyl and R₄ maybe CCl₃ or CF₃. In still additional embodiments, the R₁ may be o-anisoleand R₄ may be NO₂. In further embodiments, the R₁ and R₂ are twonaphthyl groups fused to the indole ring. Additional preferredembodiments are contemplated in which R₁ is phenyl and R₂ is CH₃.

In those aspects of the invention in which the inhibitor is a urea, theurea may have the general formula:

wherein R₁ is OR, Br, Cl, or F, R₂ is OR, NHCO₂ R, Cl, F, or H, R₃ isCl, Br, OR, or CO₂R, R₄ is Cl or Br, R₅ is H, R₆is H, R₇ is H, R₈ is aconjugated or aromatic system. R₉ is H, OR, Cl or Br, R₁₀ is H, OR, orCl. More particularly, R₁ may be OMe, and either R₃ or R₄ may be Cl, inaddition to R₈ being C(═O)Ph or a fused aromatic ring at R₇ -R₈.

In those embodiments in which the inhibitor is an aromatic amide, theinhibitor may have the general formula:

wherein R₁, R₄ and R₅ are H, R₂ and/or R₃ are small electron-withdrawinggroups, and R₆ is a substituted or unsubstituted alkyl of at least sixatoms including O, N or S, with or without a phenyl ring. Moreparticularly, the electron-withdrawing group is selected from the groupconsisting of Cl, and F. In other preferred embodiments, the R₄ and R₆in the aromatic amide of structure III are smaller conjugated systems of2-6 atoms of C, O, N or S, and includes a phenyl ring.

In those embodiments in which the inhibitor is a quinoline, theinhibitor may have the general formula:

wherein R₂ may be 3, 4-dimethoxybenzene or p-toluene, R₃ is H, R₄ may beCO₂R, C(═O)NH2, or H , R₅ is H, R₆ is H , NO₂, SO₃H, NH₂, CF₃ or CCl₃,R₇ is an alkyl group, NO₂, SO₃H, NH₂, CF₃ or CCl₃ and R₈ is H. Inparticular, the combination where R₂ is 3, 4-dimethoxybenzene, R₃ is H,R₄ is CO₂R , R₅ is H, R₆ is H , R₇ is Me, and R₈ is H.

It is particularly contemplated that the bacterium is Streptococcuspneumonia, Enterococcus faecalis, Staphylococcus aureus, Streptococcuspyogenes, Escherichia coli, Pseudomonas aeruginosa, Staphylococcusepiderimis, Mycobacterium smegmatis and Serratia marcesens. Of course,those of skill in the art will realize that the inhibitors found to beuseful in applications against these bacteria also may be useful againstother bacterial infections. As such these are exemplary bacteria and thepresent invention is not intended to be limited to infection caused bythese bacteria.

Another aspect of the present invention provides an indole having thegeneral formula:

wherein R₁ is phenyl, 2-naphthyl, o-anisole, R₂ is H or CH₃, R₁ and R₂are two naphthyl groups fused to the indole ring, R₃ is H, R₄ is NO₂,SO₃H, NH₂ and CF₃ or CCl₃, R₅ is H , and R₆ is H. In specificembodiments, R₁ is phenyl and R₄ is SO₃H or NO₂. In other embodiments,R₁ is 2-naphthyl and R₄ is CCl₃ or CF₃. In still additional embodiments,R₁ is o-anisole and R₄ is NO₂. Other embodiments contemplate an indolein which R₁ and R₂ are two naphthyl groups fused to the indole ring. Yetanother indole molecule contemplated is one in which R₁ is phenyl and R₂is CH₃.

Also contemplated herein is a urea having the general formula:

wherein R₁ is OR, Br, Cl, or F, R₂ is OR, NHCO₂R, Cl, F, or H, R₃ is Cl,Br, OR, or CO₂R, R₄ is Cl or Br, R₅ is H, R₆ is H, R₇ is H. R₈ is aconjugated or aromatic system, R₉ is H, OR, Cl or Br, R₁₀ is H, OR, orCl. More particularly, R₁ may be OMe, and either R₃ or R₄ may be Cl, inaddition to R₈ being C(═O)Ph or a fused aromatic ring at R₇-R₈.

Also contemplated herein is an aromatic amide having the generalformula:

wherein R₁, R₄ and R₅ are H; R₂ and/or R₃ are small electron withdrawinggroups, and R₆ is substituted or unsubstituted alkyl of at least sixatoms including C, O, N or S, with or without a phenyl ring.Specifically the aromatic amide may be one in which R₄ and R₆ aresmaller conjugated systems of 2-6 atoms of C, O, N or S, and includes aphenyl ring.

Another aspect of the present invention provides a quinoline having thegeneral formula:

wherein R₂ may be 3, 4-dimethoxybenzene or p-toluene, R₃ is H, R₄ may beCO₂R, C(═O)NH2, or H, R₅ is H, R₆ is H, NO₂, SO₃H, NH₂, CF₃ or CCl₃, R₇is an alkyl group, NO₂, SO₃H, NH₂, CF₃ or CCl₃ and R₈ is H. Inparticular, the combination where R₂ is 3, 4-dimethoxybenzene, R₃ is H,R₄ is CO₂R, R₅ is H, R₆ is H, R₇ is Me, and R₈ is H.

Another aspect of the present invention contemplates a method ofscreening for inhibitors of NorA comprising providing a cell expressingonly a single functional transporter, said transporter being Nor A;contacting said cell with a transportable element in the presence of acandidate inhibitor substance; and comparing the transport of saidelement by said cell with the transport of said element in the absenceof said candidate inhibitor substance.

In particularly preferred embodiments, the cell is a bacterial cell. Inadditional preferred embodiments, the bacterial cell is a Gram negativebacterial cell. In other preferred embodiments, the bacterial cell is aGram positive bacterial cell. More particularly, the Gram positivebacterial cell is a Bacillus subtilis cell. In specific embodiments, itis contemplated that the B. subtilis cell contains disrupted Bmr and Bltgenes.

In other preferred embodiments it is contemplated that the NorA isStaphylococcus aureus NorA, Streptococcus pneumoniae multidrugtransporter, or Enterococcus faecalis multidrug transporter. Inparticular embodiments the transportable element is ethidium bromide. Inother embodiments, the transportable element is a fluoroquinolone.

Another aspect of the present invention provides a method for treating asubject with a bacterial infection comprising providing to said subjecta fluoroquinolone and an inhibitor of NorA, wherein said inhibitor is anindole, a urea or an aromatic amide. In preferred embodiments, thebacterium is Streptococcus pneumonia, Enterococcus faecalis,Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli,Pseudomonas aeruginosa, Staphylococcus epidermis, Mycobocteriumsmegmatis and Serratia marcesens

Also provided herein is a pharmaceutical composition comprising afluoroquinolone and an inhibitor of NorA, wherein said inhibitor is anindole, a urea or an aromatic amide. In certain embodiments, thefluoroquinolone is selected from the group consisting of Sparfloxacin,Levofloxacin, Grepafloxacin, Temafloxacin, Clinafloxacin, Bay 12- 8039,Trovafloxacin, DU6859a, Sarafloxacin. In addition to thefluoroquinolones, it is contemplated that other quinolones such asfluoronaphthyridones may be useful in the compositions of the presentinvention. A particularly preferred quinolone is LB20304. Of course, oneof skill in the art will realize that there will be other antibacterialfluoroquinolones that may be combined with the inhibitors of the presentinvention. As such, the present invention is not limited for use incompositions with the listed fluoroquinolones alone, rather theinhibitors will be useful in combination with any fluoroquinolone orother agent that possesses antibacterial activity. Additionally, theinhibitors of the present invention will be useful with anyantibacterial agent which is or would be effective at killing, reducingor otherwise diminishing the growth of bacteria but for the presence ofresistance created by the multidrug transporters in such bacteria.

Another aspect of the present invention describes a method of enhancingthe antifungal action of azole antifungal agents comprising contacting afungus with an inhibitor of a fungal multidrug transport protein,wherein said inhibitor is an indole, a urea, an aromatic amide or aquinoline. More particularly. the indole has the general formula I, theurea has the general formula II, the aromatic amide has the generalformula III and the quinoline has the general formula XV. It isparticularly contemplated that the fungus is from a species selectedfrom the giroup consisting of Candida, Cryptococcus, Blastomyces,Histoplasma, Torulopis, Coccidioides, Paracoccidioides and Asperugillis.Of course one of skill in the art will realize that the invention is notlimited to only treating these fungal infections but rather that theinhibitors will likely be useful against many other fungal species.

Yet another embodiment of the present invention provides a method ofscreening for inhibitors of a fungal multidrug transporter comprising:providing a cell expressing only a single functional transporter, saidtransporter being fungal multidrug transporter; contacting said cellwith a transportable clement in the presence of a candidate inhibitorsubstance; and comparing the transport of said element by said cell withthe transport of said element in the absence of said candidate inhibitorsubstance. In specific embodiments, the cell is a fungal cell.

In specifically preferred embodiments, the cell is from the Candidaspecies. In other preferred embodiments, the multidrug transporter is aCandida multidrug transporter. In certain embodiments, the antifungalagent is a triazole antifungal agent. In other preferred embodiments,the triazole is selected from the group consisting of ketoconazole,miconazole, itraconazole, fluconazole, griseofluconazole, clotrimazole.econazole, terconazole and butaconazole. It should be understood thatthese triazole anti-fungal agents are exemplary agents, additionalazoles also may be useful in the present invention. Such additionalazoles may be derived form these azoles listed or have a similar mode ofaction to these compounds.

Another aspect of the present invention provides a method of treating asubject with a fungal infection comprising providing to said subject anazole antifungal agent and an inhibitor of a fungal multidrug transportprotein, wherein said inhibitor is an indole, a urea, an aromatic amideor a quinoline. In specific embodiments, the antifungal agent isselected from the group consisting of ketoconazole, miconazole,itraconazole, fluconazole, griseofluconazole, clotrimazole, econazole,terconazole and butaconazole.

Also provided herein is a pharmaceutical composition comprising an azoleantifungal agent and an inhibitor of a fungal multidrug transporter,wherein said inhibitor is an indole, a urea, or an aromatic amide.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESICRIPTION OF THE DRAWINGS

FIG. 1. CoMFA contour map for the indole sterie field. INF55 is picturedwithin this field. Green areas indicate favored regions of bulk andyellow indicates unfavorable regions for bulky groups.

FIG. 2. CoMFA contour map for the indole electrostatic field. INF55 ispictured within this field. Red areas indicate favored regions ofnegative charge and blue indicates favored regions of positive charge.

FIG. 3. CoMFA contour map for the biphenyl urea steric field.INF271,276, is pictured within this field. Green areas indicate favoredregions of bulk and yellow indicates unfavorable regions for bulkygroups.

FIG. 4. CoMFA contour map for the biphenyl urea electrostatic field.INF271, 276 is pictured within this field. Red areas indicate favoredregions of negative charge and blue indicates favored regions ofpositive charge.

FIG. 5. CoMFA contour map for the aromatic amide steric field. INF240 ispictured within this field. Green areas indicate favored regions of bulkand yellow indicates unfavorable regions for bulky groups.

FIG. 6. CoMFA contour map for the aromatic amide electrostatic field.INF240 is pictured within this field. Red areas indicate favored regionsof negative charge and blue indicates favored regions of positivecharge.

FIG. 7. Synergy curve for the combination of INF 55 with ethidium.

FIG. 8. Effect of the lead inhibitors on ethidium efflux from NA cells.NA cells were loaded with ethidium in the presence of reserpine andallowed to efflux in the absence of reserpine (A) in the presence of 20μg/ml reserpine (B), 5 μg/ml of INF 55 (C), 5 μg/ml of INF 240 (D), 5μg/ml of INF 271 (E), 5 μg/ml of INF 392 (F), or 10 μg/ml of INF 277(G). Fluorescence intensity is proportional to the amount of ethidiumremaining inside the cells.

FIG. 9. Effect of the lead inhibitors on the susceptibility of wild typeS. aureus (SA1199) to ciprofloxacin. Cells were diluted to an OD₆₀₀ of0.01 into tubes with LB medium containing different concentrations ofciprofloxacin (1.5 fold dilutions) and no inhibitor(A), 20 μg/mlreserpine (B), 5 μg/ml INF 55 (C), 5 μg/ml INF 240 (D) 5 μg/ml INF 271(E), 1.25 μg/ml INF 277 (F), 1.25 μg/ml INF 392 (G). Optical densitieswere determined after 3 h incubation.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The development of clinically useful inhibitors of the multidrug-effluxtransporters in Gram positive pathogenic bacteria, Staphylococcus aureusand Streptococcus pneumoniae, is essential if these opportunistic Grampositive infections are to be effectively treated. As stated above, Grampositive infections are notoriously difficult to treat. One majorimpediment to the effective treatment of Gram positive infections isantibiotic resistance that is mediated by multidrug transporters. Thesetransporters are involved in both intrinsic and acquired resistance tofluoroquinolone antibiotics. Staphylococci and Pneumococci, twopathogens of enormous clinical importance, are particularly retractileto fluoroquinolone therapy. Further, it is known that fungal pathogensalso have multidrug transporters that share significant homology withNorA. The present application demonstrates that inhibition of themultidrug transporters in bacterial pathogens would dramaticallyincrease the effectiveness of fluoroquinolone therapy by both increasingthe intrinsic susceptibility of these pathogens to fluoroquinolones andsuppressing the emergence of drug-resistant variants. Furthermore, theinhibitors identified herein as active against NorA also are likely toshow cross reactivity with fungal multidrug transporters and proveuseful in potentiating the antifungal effects of azole antifungal agentsby decreasing intrinsic or acquired azole resistance.

The present invention shows that the use of fluoroquinolones incombination with an inhibitor of multidrug transporters dramaticallyimproves the antibacterial efficacy of these antibiotics by bothreducing their effective concentration several fold (shifting it wellbelow their practically achievable tissue levels) and preventing theemcrgence of drug-resistant variants. These inhibitors also may beuseful in antifungal applications.

Prior to the present invention, reserpine was the only known inhibitorof bacterial multidrug transporters. Unfortunately, reserpine cannot beused to potentiate fluoroquinolones because of its neurotoxicity at therequired concentrations. The inventors have demonstrated the feasibilityof developing alternative inhibitors and identified a number ofstructurally diverse lead compounds that are highly active againstmultidrug transporters of both S. aureus and S. pneumoniae. Thiscross-species activity of the newly identified inhibitors is veryencouraging. In the majority of clinical cases, e.g., pneumonia, otitismedia, etc. physicians frequently are forced to treat patients withoutknowing the biological nature of a pathogen. The present inventionprovides an array of powerful broad spectrum inhibitors of potentiallyvery broad clinical usefulness.

1. The Present Invention

The inventors have screened a library of synthetic chemicals andidentified several promising lead compounds that effectively inhibit theS. aureus multidrug transporter NorA. Some of these lead compounds alsowere found to be effective against the presently unidentified multidrugtransporter of Streptococcus pneumoniae.

A library of compounds was screened and 399 compounds were suggested aspotential inhibitors. Of these 399, 54 showed activity at 5 μg/mL orless, while the others showed moderate to little activity at 10-20μg/mL. Three of the most potent compounds are shown below as INF55 withan indole moiety; a urea compound, INF271; and INF240, possessing anaromatic amide functional group. Since it is unclear whether NorA hasmore than one potential binding site, the compounds were subdivided intothe three groups: indoles (nitroindoles), the ureas, and the aromaticamides. These three classes of compounds were evaluated using theactivity data provided, and the CoMFA fields generated to see if a3D-QSAR relationship was present.

Structures of IFN55, INF271 and INF240

Using the insights gained from the analyses performed, the presentinvention provides methods for enhancing the antibacterial action offluoroquinolones comprising contacting bacteria with an inhibitor ofNorA in combination with the fluoroquinolone therapy. The inhibitor maytherefore be an indole, a urea or an aromatic amide. More particularly,the indole will have a generic formula (I) in which R₁ is phenyl,2-naphthyl or o-anisole, R₂ is H, or CH₃, R₁ and R₂ are two naphthylgroups fused to the indole ring, R₃ is H, R₄ is NO₂, SO₃H, NH₂ and CF₃or CCl₃, R₅ is H, and R₆ is H.

In a particular example of the indole used in the present invention, R1is a phellyl group and R₄ is an SO₃H group (structure IV); in anotherexample the indole has a phenyl group at R₁ and an NO₂, group at R4(structure V). Structure VI shows an indole of the present invention inwhich R₁ is 2-naphthyl and R₄ is CCl₃, the indole of structure VII has2-naphthyl at R₁ and CF₃ at R₄. Structure VIII shows an indole of thepresent invention in which R₁ is o-anisole and R₄ is NO₂. Yet anotherindole of the present invention has a naphthyl groups fused to theindole rings (structure IX). Also contemplated to be useful in thepresent invention is a structure wherein R₁ is phenyl and R₂ is CH₃(structure X), a more particularly defined indole having this structureis shown in structure XI, in which R4 is further defined as an NO₂group. Of course these are exemplary indoles of the present invention,additional indoles may be useful as and described herein.

In specific embodiments of the present invention the inhibitor is a ureahaving the general formula:

wherein R₁ is OR, Br, Cl, or F, R₂ is OR, NHCO₂R Cl, F, or H, R₃ is Cl,Br, OR, or CO₂R, R₄ is Cl or Br, R₅ is H, R₆ is H, R₇ is H, R₈ is aconjugated or aromatic system. R₉ is H, OR, Cl or Br, R₁₀ is H, OR, orCl. More particularly the present invention contemplates a biphenyl ureawhere R₁ is OMe, and either R₃ or R₄ may be Cl, in addition to R₈ beingC(═O)Ph, a fused aromatic ring at R₇-R₈, or a Ph at R₇. In otherexamples, R₁ is OMe, R₄ is Cl, and R₉ is OR, Br, or I. Certain examplesalso have a urea (II) with R₂ and R₃ being Cl, and R₈ being C(═O)Ph, orR₁ and R₄being Cl, or R₉, being OR.

In specific embodiments, the inhibitor is an aromatic amide that has thegeneral formula:

wherein R₁, R₄ and R₅ are H, R₂ and/or R₃ are smallclectron-withdrawingy groups, and R₆ is substituted or unsubstitutedalkyl of at least six atoms including O, N or S, with or without aphenyl ring. More particularly, the electron-withdrawing group may be aCl or an F moiety. Other aromatic amides of the present invention havesmaller conjugated systems of 2-6 atoms of C, O, N or S at R₄ and R₆,and include a phenyl ring.

In other embodiments, the inhibitor is a quinoline that has the generalformula:

wherein R₂ may be 3, 4-dimethoxybenzene or p-toluene, R₃ is H, R₄ may beCO₂R, C(═O)NH2, or H, R₅ is H, R₆ is H, NO₂, SO₃H, NH₂, CF₃ or CCl₃, R₇is an alkyl group, NO₂, SO₃H, NH₂, CF₃ or CCl₃, and is H₈. Inparticular, the combination where R₂ is 3, 4-dimethoxybenlzene, R₃ is H,R₄ is CO₂R, R₅ is H, R₆ is H, R₇ is Me, and R₈ is H.

The present specification shows that the above compounds are useful incombinations with fluoroquinolones in the treatment of bacterial andmore particularly Gram positive bacterial infections. These compoundsalso may be useful in anti-fungal applications. Method and compositionsfor the production and/or screening for the activities of thesecompounds are discussed in further detail herein below. Similarly, thesecompounds may be used as lead compounds for generating additionalcompounds that will be useful as inhibitors of multidrug transporters inbacterial and fungal pathogens.

2. Drug Efflux Proteins in Multidrug Resistant Bacteria

Bacteria contain an array of transport proteins in their cytoplasmicmembrane. Many of these proteins play an important role in conferringintrinsic and acquired resistance to toxic compounds. Severalchromosomally encoded multidrug transporters have been identified inGram positive bacteria including Bmr (Neyfakh et al., 1991). Blt (Ahmedet al., 1995), Bmr3 (Ohki and Murata, 1997) in Bacillus subtilis, NorAin Staphylococcus aureus (Neyfakh et al., 1993; Yoshida et al., 1990),LmrP (Bolhuis et al., 1995) and LmrA (van Veen et al., 1996) inLactobacillus lactis and LfrA in Mcyobacteriaum smegmatis (Takiff etal., 1996).

One of the most effective regimens for controlling Gram negativeinfections employs fluoroquinolone compounds. One Such compound,ciprofloxacin (Davis et al., 1996) accounts for 90% of all quinolonesused in medicine (Acar and Goldstein, 1997). Because of its spectrum ofactivity, oral availability, and relatively low cost. ciprofloxacin hasbeen used for treating a wide range of infections, including those ofunknown etiology. Although it is highly active against most Gramnegative microorganisms (MIC₉₀ in the range of 0.1 μg/ml), ciprofloxacinis much less effective against Gram positive infectious. particularlyaerobic Gram positive cocci. The MIC₉₀ values for S. aureus, E. faecalisand S. pneumoniae are in the range of 1-5 μg/ml, whereas the achievabletissue concentration of ciprofloxacin is only 4 μg/ml (Davis et al.,1996). The high intrinsic resistance to ciprofloxacin and the extensiveuse of quinolones both in human and veterinary medicine has led to theemergence and dissemination of ciprofloxacin-resistant Gram-positivestrains. This resistance is thought to be due to the presence ofspecific multidrug transporters in the Gram positive bacteria.

In addition to being involved in the intrinsic resistance ofGram-positive cocci to fluoroquinolones, multidrug transporterscontribute to the acquired resistance, which is selected upon exposureto these antibiotics. In S. aureus and S. pneumoniae, the acquiredresistance has so far been attributed mainly to the sequentialacquisition of mutations in the targets of fluoroquinolone action,topoisomerase IV and DNA gyrase (Cambau and Gutman, 1993; Ferrero etal., 1994; Munoz and De La Campa, 1996; Tankovi. 1996). From the limitedstudies of fluoroquinolone resistance mechanisms in E. faecalis, itappears that mutations of gyrase are present in at least some high levetresistant isolates Korten et al., 1994). However, it has become apparentin recent years that these mechanisms of acquired resistance arecomplemented by over-expression of multidrug transporters. Suchover-expression can result from either amplification of the transportergene (Neyfakh, 1991); or mutations in the regulatory regions of thesegenes or regulatory proteins controlling their transcription (Ahmed etal., 1995; Kaatz and Seo., 1995).

NorA is a multidrug transporter involved in both the intrinsic andacquired resistance of the pathogen Staphylococcus aureus to a varietyof unrelated compounds including a number of widely used fluoroquinoloneantibiotics, by means of their active extrusion from the bacterial cell.ihe present invention identifies and characterizes numerous inhibitorsof NorA.

The inventors' recent studies indicate that a multidrug efflux mechanismalso appears to contribute to the intrinsic and acquired fluoroquinoloneresistance of Streptococcus pneumoniae, another clinically importantGram positive pathogen which has only a moderate susceptibility tociprofloxacin (MIC₉₀ 1-2 μg/ml). The present invention shows that someof the most active NorA inhibitors are also effective in promoting,ciprofloxacin bacteriotoxicity in S. pneumoniae. The inventors suggestthat the lead inhibitors will also be effective in promotingfluoroquinolone bacteriotoxicity, not only in S. aureus, but also in S.pneumoniae and E. faecalis. The following section summarizes the recentfindings supporting the involvement of a multidrug efflux mechanism inthe ciprofloxacin resistance of S. pneumoniae.

The present inventors recently reported the presence of anefflux-dependent fluoroquinolone resistance mechanism in S. pneumoniaeselected for increased resistance to ethidium bromide (Baranova andNeyfakh, 1997). Ethidium resistance in the selected strain, called EBR,was shown to result from increased efflux of this drug. EBR alsodemonstrates increased resistance to the fluoroquinolones ciprofloxacinand norfloxacin, suggesting the contribution of a multidrug effluxtransporter, tentatively termed PmrA. Although no cross resistance ofthis strain to the Bmr and NorA substrate rhodamine was observed,reserpine, at non-toxic concentrations, inhibited ethidium efflux andreversed the resistance to both ethidium and fluoroquiniolones.Furthermore, reserpine was shown to potentiate the susceptibility ofwild type S. pneumoniae to ethidium and fluoroquinolones by two to threefold. This suggests that, like other multidrug transporters in Grampositive bacteria, this efflux mechanism may contribute to the intrinsicand acquired resistance of S. pneumoniae to fluoroquinolone antibiotics.

Analogous to S. aureus, mutations in topoisomerase IV precede mutationsin gyrase in stepwise selected ciprofloxacin-resistant mutants of S.pneumoniae (Tankovic et al., 1996). However, unlike S. aureus, thereappears to be an additional stage that precedes the acquisition ofmutations in topoisomerase IV, namely, selected cells demonstrateelevated fluoroquinolone resistance with no detectable mutations in thetopoisomerase or gyrase genes (Tankovic et al., 1996). The inventorsspeculated that fluoroquinolone resistance at this stage may result fromthe increased expression of the putative multidrug efflux transporterPmrA, and thus, not only would such mutants exhibit cross resistance toethidium bromide but also that reserpine would decrease their augmenteddrug resistance. To investigate this possibility, the inventors selectedin vitro first step mutants of S. pneumoniae (ATCC 49619), resistant tofour-fold the MIC of ciprofloxacin (2 μg/ml). Selection of 10⁹ cellsyielded fifteen such mutants, of which three were analyzed further.Compared to the parental strain, all three mutants exhibited aneight-fold increase in the ciprofloxacin MIC. Interestingly, the MIC ofethidium bromide for these three mutants also increased, by 8-16 fold.Furthermore, a non-toxic concentration of reserpine reversed theresistance to both ciprofloxacin and ethidium bromide. Similar to theEBR strain, no increase in resistance to rhodamine was observed,suggesting the involvement of the same transporter, PmrA.

These data indicate that a multidrug efflux transporter not onlycontributes to the intrinsic fluoroquinolone susceptibility of S.pneumoniae, but also mediates resistance to fluoroquinolones in firststep mutants of this pathogen. Supporting this notion, Zeller et al.(1997) recently reported that a first step in vitro selectedciprofloxacin resistant mutant of S. pneumoniae which had no alterationsin Topo IV, exhibited increased efflux of ciprofloxacin and a drugresistance profile resembling that conferred by NorA expression.

Multidrug transporters also play an important role in both the intrinsicand acquired resistance of important fungal pathogens to antifungalagents. Particularly, multidrug transporters contribute to theresistance of Candida albicans, the fourth leading cause of allhospital-acquircd infections, to azolc antifungal agents. A number ofthese fungal multidrug transporters belong to the major facilitatorsuperfamily of membrane transporters and share significant homology withNorA. The inhibitors identified as active against NorA are highly likelyto show cross reactivity with fungal multidrug transporters and proveuseful in potentiatinig the antifuingal effects of azole antifungalagents by decreasing intrinsic or acquired azole resistance.

3. Chemical Synthesis of Diverse Analogs of the Lead Inhibitors.

As described elsewhere herein, the inventors have a substantial databaseof compounds with varying multidrug transporter inhibitory activities.It is particularly intriguing that in at least a few cases, the shift ofa bond by one position on an aromatic ring can substantially diminishthe activity of some of the most potent inhibitors, suggjesting thatthere are very specific structural requirements for binding andinhibition. The availability of extensive structural information on bothactive and inactive analogs will provide a high quality analysis of theeffect of various structural variations upon activity.

Many of these inhibitors are highly flexible, making it impractical toperform conventional “2D” QSAR analysis. However, recent techniques havebeen developed that permit classification of compounds by 3D geometry,and decomposition of activities into various “molecular field” effects.The DISCO (Tripos) and CoMFA (Tripos) software was used for theseanalyses. Thus, the goal of this analysis was to determine thosestructural features of the various inhibitors that are most effective inenhancing binding specificity, with emphasis on the three-dimensional or“topological” relationship amongst critical pharmacophores.

As stated earlier, the most active inhibitors fall into severalchemically distinct classes suggesting that there may be multiplebinding modes, with different chemical structures binding to partiallyor completely distinct sites within the efflux protein. However, throughDISCO analysis of the structure-activity patterns, it is feasible tocluster compounds that bind in a similar mode, and distinguish betweenclusters that bind in distinctly differing manners (Martin et al.,1993). Using this strategy, it is possible to evaluate the probabilityof the various chemical classes binding in physically distinct modes. Itshould be noted that, while there are differing “core” chemicalstructures, the similarity of some of the “external” moieties suggeststhat there may also be similarities in the three-dimensional topologies.

INF 55 and INF 271 were used as a pharmacophore model for predictingfurther analogs with higher activity, because the inventors' initialdata indicated that these two inhibitors are both highly effectiveinhibitors, and appear to bind in modes for which the development ofresistance is nearly minimal. The pharmacophore models for these and theinventors' other high activity inhibitors are then be used as guidancein the synthetic strategies outlined schematically below.

The present section, therefore provides details of conventional chemicalsynthesis strategies in the development of second generation inhibitoranalogs for both scientific and economic reasons. At this stage, theinventors have several lead compounds that will require somewhatdiffering synthetic strategies for analog development. These leadcompounds fall into three broad classes of indoles, ureas and aromaticamides. The synthetic strategies in each of these class is discussed inthe present section.

a. Indoles

The initial screening process identified a series of nitroindolederivatives. The most potent of these are compounds 1-6 whose activitydecreases in the order 1>2˜3>4˜5˜6.

These six compounds may be broadly summarized as having i) an electronwithdrawing nitro group in the benzene ring of the indole moiety, andii) a lipophilic alkyl or aryl group attached to position 2- or 3- orboth of the heterocyclic ring. A further series of indoles with eitheran electron donating group on the benzene ring and/or a polarheteroatomic based side chain on the heterocyclic ring were considerablyless active or even inactive. All of the active indoles have a freeindole NH group. Thus, in this series of compounds it would seem mostappropriate to undertake a systematic study of rational analogs of thegeneral class represented by formula (7). The synthesis proceeds in astepwise fashion. Firstly, a further series of 2 and 3-alkyl or arylindole derivatives all retaining the 5-nitro substituent aresynthesized. Subsequentlyv with the optimum alkyl group and location, itis possible to systematically vary the nature and position of theelectron withdrawing group.

i. Optimization of the Lipophilic Stibstituents

Judicious combination of structures 1-4 suggests that compounds 9 and 10should have a high priority for investigation. Neither 9 nor 10 areknown compounds but the corresponding derivatives in which the nitrogroup is replaced by a proton are both known, and have been prepared bythe Fischer indole synthesis (Robinson, 1982; Kulagowski et al., 1985;Katritzky and Wango 1988). This gives the inventors a high degree ofconfidence that both 9 and 10 will be available in one step by Fischerindole reaction of 4-nitrophenylhydrazine with α- and β-tetralone,respectively (Schemes 1 and 2). All of these starting materials areavailable commercially. The inventors expect that 11 will be a minorproduct in the synthesis of 10; it will be readily separated from 10chromatographically, easily distinguished by routine NMR spectroscopyand, of course, screen for activity.

It is likely that the phenyl and indole ring in both compound 1 andcompound 3 adopt a non-planar conformation for example as shown incompound 13 to minimize steric interactions. If this is indeed the caseand if the major conformation is the bound one, models 10 and 11 will beless than ideal as they will hold the two aryl groups close to coplanar.In order to test for this possibility higher homologs (compounds 14-17)of compound 10 and compound 11 are synthesized, again by the Fischerindole synthesis replacing α- and β-tetralones by the corresponding,readily available benzocyclohepotanones and benzocyclooctanones. In thisseries, as n increases from 1 to 2 to 3 the torsion angle between thetwo ringis will increase enabling probing of the optimal conformationfor binding. The maximum torsion angle (90°) will be best probed via anopen chain system such as in compound 18. Again this system isaccessible by Fischer indole synthesis, using 2-methylpropiophenone asthe ketone component. In addition to this series of constrained 2- and3-phenyl indole analogs, aliphatic substituents at positions 2 and 3-are sufficient may also be useful; indeed the activity of compound 2 andcompound 5 suggests that this may be the case. Such a series ofcompounds in which the bulk of the alkyl groups is systematicallyincreased also is readily accessible by the Fischer indole synthesis.For example the regiosiomers 19 and 20 are prepared by condensation of4-nitrophenylhydrazine with pinacolone (tert-butyl methyl ketone) and2-tert-butylacetaldehyde, respectively.

The preparation of each of the above 4-nitroindoles is extremelystraightforward and takes place in a single step from4-nitrophenylhydrazine and a simple ketone. Moreover most of the ketonesrequired are commercially available. Those which are not availablecommercially are all known compounds for which short, simplepreparations are described in the literature. Thus, one of skill in theart will be able to prepare and purify these compounds in sufficientquantity for screening. From these compounds, it is then expected thatfurther alkyl and aryl combinations may be assayed, subsequent to theinitial phase.

ii. Location and Nature of the Eectron Withdrawing Group

Having established the optimum combination of alkyl and or aryl groupsat the 2-and 3- positions, the best location for the polar group in thebenzene ring is determined. This involves a relatively straightforwardprocess, since, in addition to p-nitrophenylhydrazine already employedfor the 5-nitro derivatives, o- and m-nitrophenyl hydrazine arecommercially available compounds. The syntheses are illustrated inSchemes 3 and 4 with acetophenone as an exemplary ketone, as this willlead to regioisomers of the 2-phenyl-5-nitro compound (compound 1) whichwas the most active inhibitor from the initial phase of theinvestigations. However, it is understood that the ketone leading to theoptimum selection of hydrophobic groups, as determined from thesynthesis in Schemes 1 and 2 above, will be employed in practice inthese routine indole syntheses.

With m-nitrophenylhydrazine the synthesis leads to a mixture of the 4-and 6-nitroindoles i.e., compound 21 and i.e., compound 22, respectively(scheme 3). These compounds are separated using standard chromatographictechniques. Subsequent NMR spectroscopy allows the designation of theappropriate structures to the separated compounds. The 7 nitroindolederivative compound 23 is prepared using o-nitrophenylhydrazine as shownin scheme 4.

Finally, the inventors turned to the nature of the electron withdrawinggroup. The lead compounds for this synthesis is one which presents theoptimum inhibitory activity from the compounds of lipophilic groups inthe heterocyclic ring and for the optimum location in the benzene ringboth as determined above. The following provides an illustration of thechemistry for the synthesis of 2-phenylindole with an electronwithdrawing group in the 5-position, as in the lead compound 1.Likewise, any other regioisomer that has proves optimal inhibitoryactivity as described herein may be derivatized as outlined in Schemes 3and 4.

Alternatively wherever the appropriately substituted hydrazine iscommercially available the inventors subject it to the Fischer indolereaction. This was the case with 4-fluor and 4-methylsulfonyl hydrazine(scheme 5). The inventors note that 2- and 3-fluorophenylhydrazine alsoare commercial, which means that the regioisomeric indoles will beavailable with a minimum of effort should they be required.

In some instances, it will prove convenient to synthesize the hydrazonenecessary for the Fischer indole reaction from the correspondingsubstituted aniline derivative. For example this approach may beconvenient for the trifluoromethyl substituted series (scheme 6) becauseo-, m-, and p-trifluoromethylaniline are all readily commerciallyavailable.

Similarly, the same reaction format may be used to prepare 2-phenyl5-iodoindole (or its regioisomers) which will subsequently be benzylatedon the indole nitrogen to give the derivative compound 27 (scheme 7).

Compound 27 may be transmetallated with tert-butyllithium to give the5-litho derivative compound 28 which in turn will serve for theintroduction of carboxylic (compound 29), sulfonic (compound 30). andphosphonic acid (compound 31) derivatives. Each of these couplings willrequire a final deprotection of the N-benzyl indole by hydrogenolysis(scheme 8).

b. Urea Compounds

In the initial screening the inventors isolated three urea compounds(32-34) having high inhibitory activity, but with considerablestructural variation. The optimization of these urea leads is describedin detail herein below.

The chemistry of ureas is relatively straightforward and the potentialfor such compounds in medicinal chemistry is very well established.Nowhere is this better highlighted than with the extremely successful,urea based-protease inhibitors introduced in the last few years for thetreatment of HIV. The urea-based protease inhibitory compounds areconsiderably more complex than the structures envisaged here, yet thechemistry of urea synthesis is such that they may be producedcommercially on a very large scale. The most straightforward synthesisof unsymmetric ureas involves the condensation of a first primary aminewith phosgene to give an isocyanate, which is subsequently used tocapture a second amine (scheme 9). Numerous or(ganic synthesis protocolsare available for this type of reaction.

One common factor in the lead compounds 32-34 is the presence of atleast one apolar aromatic substituent, i.e., 4-chlorophenyl in compound32; 2-naphthyl in compound 33; and 3,4-dichlorophenyl in compound 34.The commercial availability of 4-chlorophenyl isocyanate (compound 35)makes it a suitable candidate to be selected as the starting point forthe semi-systematic approach to optimization. Thus, compound 35 iscondensed with a wide selection of commercial primary aromatic andaliphatic amines to give the ureas such as compound 36 (scheme 10). Theamines R′—NH₂ may be selected to probe the essential requirements of the“right hand side” group (R′) of the target ureas. Thus it can bedetermined whether an aryl or alkyl group is preferred and ifelectron-donating or electron withdrawing functional groups areadvantageous and, if so, at what site.

Once an optimal R′ group is located for the “right hand side” of themolecule its precursor amine R′—NH, (compound 37) is converted to thecorresponding isocyanate, compound 39, which is then be condensed withthe same selection of primary aromatic and aliphatic amines, now asR″—NH₂, to probe the optimal requirements for the left hand side group(R″) in compound 40 (Scheme 11). Depending on the functionality presentin the optimal amine R′—NH (compound 37) it can be converted to anisocyanate compound 39 with phosgene or with carbonyl imidazolide(compound 38). Phosgene is used for rapidity and ease of purificationwhen R′ is a simple aliphatic or aromatic amine devoid of othernucleophilic centers, whereas its milder, more discriminating analog 38(Staab and Benz, 1961) is employed with more sensitive R′ groups.

In this manner, for a purchase of 4-chlorophenylisocyanate (compound 35)and fifty commercial primary amines, it is possible to synthesize aninitial group of fifty ureas (36). Taking the optimal R′ group (compound37) from this selection, converting it to the isocyanate 39, and thencondensing with the same fifty amines will yield a second generationseries of fifty ureas 40 from which an advanced lead urea compound maybe selected and the more precise requirements for both R′ and R″determined.

c. 2,5-Disubstituted Pyrimidine-4,6-diones

The most active lead compound in this category was the thiouracilderivative compound 41. A more general structure is represented by theformula compound 42. In order to probe the requirements of thepharmacophore in terms of the two hydrophobic substituent groups, adiverse range of compounds 42 are synthesized in which R and R′ aresystematically varied. The symmetry inherent in compound 42 and itsimmediate precursor compound 43 suggest that these compounds will bemost readily accessed by a common variant on the Principal Synthesis ofpyrimidines. This very well established chemistry is extensivelydocumented in a recent (1994) encyclopedic compilation of the pyrimidineliterature (Brown, 1994).

Thus, as indicated in Scheme 12, a range of diethyl alkylmalonate esters44 may be condensed with thiourea in the presence of sodium ethoxide togive compound 43. This variant on the Principal Synthesis is very wellestablished for all classes of alkyl group (R′) and numerous examplesare given in the recent review (Brown, 1994). When R′ is a primary orsecondary alkyl group, the malonate 44 will be obtained routinely byalkylations of diethyl malonate (compound 45) as indicated in scheme 12.When R′ is tertiary alkyl, aryl, or vinyl the malonate 44 will be bestaccessed by condensation of the appropriate ester 46 with ethylchloroformate, again as indicated in scheme 12.

With 43 in hand, it is possible to turn to the elaboration of analogs of41. Here, when the desired alkyl group R is a simple primary orsecondary alkyld 43 will be alkylated in a straightforward manner withthe appropriate alkyl halide and base (Scheme 13). Again there are manyexamples of such processes in the pyrimidine literature (Brown, 1994).Neither alkylation on nitrogen, nor competing alkylation at either ofthe two oxygen atoms is reported to be problematic owing to the veryhigh nucleophilicity of such molecules on sulfur (Brown, 1994).

Heating 42 in the presence of an excess of tertiary thiol or arene thiolwill enable displacement of methanethiol and the formation ofderivatives 47 and 48, which cannot be prepared by the direct alkylationroute (scheme 14). The displacement of thiols from pyrimidines in suchnucleophilic substitution reactions is well known to those of skill inthe art (Brown, 1994).

It will be of interest to replace the sulfur atom in 42 with an oxygenor a nitrogen atom as in formulae 49 and 50. This will again be readilyachieved through the aegis of 42 (R═Me) and treatment with excessalcohol or base as appropriate (Scheme 15) (Brown, 1994). Certainly, itis more usual in pyrimidine chemistry to make use of chloride as leavinggroup in such nucleophilic displacements. The requisite chlorides are inturn prepared from the pyrimidone by treatment with POCl₃, or relatedsubstance. Unfortunately, this is not an option here as any treatment ofthe 2-oxo-analog of 42 with POCl₃ will lead to the preferentialintroduction of chlorine at the 4- and 6-positions (Brown, 1994). Thus,the chemistry advanced in Schemes 14 and 15 is designed with the dualobjective of i) minimizing effort in the laboratory by taking maximumadvantage of the readily available 42 and 43 and ii) overcoming the needfor a circuitous route for the selective introduction of chlorine at C2.

Finally, it will be important to determine whether or not it isnecessary to have a heteroatom at C2 at all. Analogs with all carbonside chains at position 2 may be prepared by another known variant ofthe Principal Synthesis (Brown, 1994) in which amidines are condensedwith the malonate esters 44, as shown in Scheme 16.

In synthesizing these analogs of 41 the inventors reason that the mostefficient approach will be to initially prepare and screen a broad rangeof compounds 42 with good diversity in the groups R and R′. Once thebetter combinations of R and R′ are identified the inventors will thentarget the synthesis of a more limited range of the syntheticallyslightly more elaborate derivatives 47-51.

d. Aromatic Amides

The preparation of aromatic amides related to INF 240 is expected to bestraightforward. Thus, for example, it will be well within the skill ofone in the art to convert ethyl aminobenzoate 52 (scheme 17), whoseortho, meta- and para-isomers are all commercially available to ittert-butyloxycarbonyl derivative 53. Controlled reduction at −78° C.with diisobutylaluminum hydride (DIBAL-H) will then provide the aldehyde54 (Jurczak et al., 1989) Wittig olefination will subsequently afford55. Conditions are available for the preferential formation of both cis-and trans-alkenes by Wittig and related olefination reactions, makingboth stereoisomers about the double bond readily available (Maryanoffand Reitz, 1989; Vedejs and Peterson, 1994). Treatment of 55 withtrifluoracetic acid will cleave the carbonate giving 56, which will thenbe coupled to an acyl chloride giving the final product 57. A furthervariation on this straightforward scheme involves catalytichydrogenation of 55, giving 58 which will then be converted through 59to 60, the saturated analog of 57. An enormous variety of acid chloridesare commercially available which will permit extensive investigation ofthe righlt-hand side of the molecule. Similarly, a considerable numberof Wittig reagents are commercially available and many others arereadily prepared by standard protocols from the corresponding alkylhalides. Thus it is expected that this reaction scheme will provideaccess to a broad cross-section of differentially substituted aromaticamides.

4. Screening

In certain embodiments, the present invention concerns a method foridentifying inhibitors of multidrug transporter proteins in bacteria.More particularly, the bacteria are Gram positive bacteria. The methodsalso concern identifying inhibitors of multidrug transporters of fungalpathogens. The multidrug transporter protein may be any protein that isinvolved with the efflux of antibacterial agents from a bacterial cellthereby contributing to drug resistance in the particular bacteria.Examples of bacterial infections that may be treated by the inhibitorsinclude but are not limited to those mediated by S. aureus, S.pneumoniae, B. subtilis E. faecalis, S. epidermidis, M. smegmatis, M.tuberculosis, and S. pyogenes. Fungal infection also may be treated bythe inhibitors of the present invention. Such fungal infections mayresults from pathogens such as Candida albicans and other Candidaspecies, as well as Cryptococcus neoformans, Blastomyces dermatitidis,Histoplasma capsulatum, Torulopis glabrata, Coccidioides immitis,Paracoccidioicdes braziliensis and Aspergillis. The present inventionthus provides methods of identifying inhibitors of multidrug transportproteins such as NorA, Bmr, Blt, Bmr3, PmrA, LmrP and LmrA. It iscontemplated that this scrcening technique will prove useful in thegeneral identification of any compound that will inhibit the efflux offluoroquinolones in multidrug resistant bacteria (or fungi) andtherefore potentiate the effects of the fluoroquinolones in the cells.

Useful compounds in this regard will not be limited to those mentionedabove. The active compounds may include fragments or parts ofnaturally-occurring compounds or may be only found as activecombinations of known compounds which are otherwise inactive. However,prior to testing of such compounds in humans or animal models, it may benecessary to test a variety of candidates to determine which havepotential.

Accordingly, in screening assays to identify pharmaceutical agents whichinhibit multidrug transporters in bacteria and fungi, it is proposedthat compounds isolated from natural sources, such as animals, bacteria,fungi, plant sources, including leaves and bark, and marine samples maybe assayed as candidates for the presence of potentially usefulpharmaceutical agents.

On the other hands one may simply acquire, from various commercialsources, small molecule libraries that are believed to meet the basiccriteria for useful drugs in an effort to “brute force” theidentification of useful compounds. Screening of such libraries,including combinatorially generated libraries, is a rapid and efficientway to screen large number of related (and unrelated) compounds foractivity. Combinatorial approaches also lend themselves to rapidevolution of potential drugs by the creation of second, third and fourthgeneration compounds modeled of active, but otherwise undesirablecompounds.

One library for the compounds identified in the present invention isDiverSet™ (ChemBridge Corp., Glenview, Ill.) The screening of thislibrary consisting of 9.600 compounds, has been completed. The chemicallibrary was screened for compounds effective, at concentrations of 20μg/ml or less, in reversing the resistance of a specially created B.subtilis strain NA to the NorA substrate ethidium bromide. Although thepresent invention employed NorA as the multidrug transporter in thescreening assays it will be understood by one of skill in the art thatthe compounds identified herein will likely have applicability to othermultidrug transporters.

In these embodiments, the present invention is directed to a method fordetermining the ability of a candidate substance to inhibitors of NorAactivity comprising generally including the steps of:

(a) providing a cell expressing only a single functional multidrugtransporter, said transporter being Nor A;

(b) contacting said cell with a transportable element in the presence ofa candidate inhibitor substance; and

(c) comparing the transport of said element by said cell with thetransport of said element in the absence of said candidate inhibitorsubstance.

To identify a candidate substance as being capable of inhibiting NorAactivity, one would measure or determine the transport of thetransportable substance (e.g., ethidium bromide) by a cell thatexpresses NorA, in the absence of the added candidate substance. Onewould then add the candidate substance to the cell and re-determine theefflux of ethidium bromide in the presence of the candidate substance. Acandidate substance which reduces the transport of the ethidium bromiderelative to the transport in its absence is indicative of a candidatesubstance with inhibitor capability. Although the present sectiondiscusses ethidium bromide as a transportable element, it is understoodthat these assays may also be performed using any fluoroquinolone whoseeffect may be monitored by bacteriotoxicity or fungicidal assays.

The candidate screening assay is quite simple to set up and perform.Thus, after obtaining a suitable test cell that has an active multidrugtransporter, one will admix a candidate substance in the presence of atransportable substance with the cell, under conditions which wouldallow the uptake of the transportable substance, for example, ethidiumbromide, an antibiotic fluoroquinolone and the like. The inhibition ofthe transporter can thus be measured by monitoring, for example growth.

In an exemplary assay, in order to identify a candidate substance as aninhibitor of NorA, the B. subtilis strain NA may be used. Cells in alogarithmic phase of growth are diluted to an OD600 of e.g, 0.002 andincubated with an effective amount of a candidate substance in thepresence of ethidium bromide, fluoroquinolone and the like (e.g., finalconcentration ¼ MIC ethidium bromide). The cells are transferred to ahumidified chamber at optimal growth conditions for an appropriateperiod of time (e. a, 37° C. for 5 hours) and subsequently examined forgrowth. Potential inhibitors of transport may be identified as thosecompounds that increase the bactericidal effect of the ethidium bromide,fluoroquinolone and the like.

“Effective amounts”, in certain circumstances, are those amountseffective at reproducibly increasing the bacteriostatic effect of theethidium bromide or fluoroquinolone in a multidrug resistant bacterialcell in comparison to the level of bactericidal activity of the ethidiumbromide or fluoroquintolone in the absence of the candidate substance.Compounds that achieve significant appropriate changes in bactericidalactivity of the fluoroquinolone will be used. Thus, a battery ofcompounds may be screened in vitro to identify other agents for use inthe present invention. Tlhe amounts of inhibitors useful in this contextmay be determined by those of skill in the art and may vary from about10 ng/ml to about 100 μg/ml. Thus it is contemplated that concentrationranges between these concentrations will be useful including but notlimited to 20 ng/ml; 40 ng/ml; 60 ng/ml; 80 ng/ml; 100 ng/ml; 120 ng/ml;140 ng/ml, 160 ng/ml; 180 ng/ml, 200 ng/ml, 350 ng/ml, 400 ng/ml, 450ng/ml, 500 ng/ml, 550 ng/ml, 600 ng/ml, 650 ng/ml, 700 ng/ml, 750 ng/ml,800 ng/ml 900 ng/ml, 1 μg/ml, 5 μg/ml, 10 μg/ml, 15 μg/ml, 20 μg/ml, 25μg/ml, 30 μg/ml, 35 μg/ml, 40 μg/ml, 45 μg/ml, 50 μg/ml, 55 μg/ml, 60μg/ml, 65 μg/ml, 70 μg/ml, 75 μg/ml, 80 μg/ml, 85 μg/ml, 90 μg/ml, and100 μ/ml

A significant increase in bactericidal (or fungicidal) activity, e.g, asmeasured using growth curve analysis are represented by a reduction inbacterial (or fungal) growth of at least about 30%-40%, and mostpreferably, by decreases of at least about 50%, with higher values ofcourse being possible. Bacterial and fungal growth assays are well knownin the art. Therefore, if a candidate substance exhibited multidrugresistance inhibition in this type of study, it would likely be asuitable compound for use in the present invention.

Quantitative in vitro testing of the inhibitor is not a requirement ofthe invention as it is generally envisioned that the agents will oftenbe selected on the basis of their known properties or by structuraland/or functional comparison to those agents already demonstrated to beeffective. Therefore, the effective amounts will often be those mountsproposed to be safe for administration to animals in another context,for example, as disclosed herein. There is considerable informationavailable on the use and doses of chemotherapeutic agents alone. whichinformation may now be employed with the present invention.

5. Fluoroquinolones

The therapeutic class of compounds known as the fluoroquinolones iswidely known and used in antibacterial treatments (U.S. Pat. No.4,448,962; DE 3,142,854, EP 206283; U.S. Pat. No. 4,499,091; U.S. Pat.No. 4,704,459; U.S. Pat. No. 4,795,751; U.S. Pat. No. 4,668,784; U.S.Pat. No. 5,532,239 each specifically incorporated herein by reference).Particularly preferred fluoroquiniolonies for use in combination withthe multidrug transport inhibitors of the present invention include butare not limited to pefloxacin, norfloxacin, ciprofloxacin, ofloxacin,sparfloxacin, grepafloxaci, Bay 12-8039, trovafloxacin, DU6859a,sarafloxacin, LB20304, levofloxacin, enoxacin, fleroxacin, lomefloxacin,temofloxacin, amifloxacin, tosufloxacin, flumequine, rufloxacin,clinafloxacin and the like. The following section describes treatmentregimens using certain fluoroquinolone, these examples merely provide anapproximation of the concentrations and formulations of fluoroquinolonesthat may be used and are not intended to be limiting in any way.

Levofloxacin is a commercially available fluoroquinolone sold under thename Levaquin™ (Ortho-McNeil). It is a synthetic broad spectrumantibacterial agent that may be formulated for intravenousadministration or for oral administration. Chemically it is a chiralfluorinated carboxyquinolone that is the S-enantiomer of the drugsubstance ofloxacin. Levaquin™ is readily available in single useinjection as well as appropriately configured solutions in premixflexible containers.

Following a single 60 minute intravenous infusion of 500 mg oflevofloxacin to healthy volunteers the peak plasma concentrationattained is 6.2 μg/ml. The plasma concentration profile of thelevofloxacin after i.v. administration is similar and comparable inextent of exposure to that observed for levofloxacin tablets when anequal (mg/mg) dose is administered. Thus, the oral and i.v. routes ofadministration can be considered interchangeable.

Levofloxacin has been shown to be active against Gram negative and Grampositive bacteria. Examples of Gram positive bacteria that levofloxacinhas been shown to be useful against include E. faecalis, S. aureus, S.pneumoniae, S. pyogens, C. perfringens, S. epidermidis, Streptococcus(Group C/F), Streptococcus (Group G), Staphylococcus saprophyticus andStreptococcus agalactiae. Gram negative bacteria shown to be inhibitedby levofloxacin include E. cloacae, E. coil, H. influenzae, Hparainfluenzae, K. pneumoniae, L. pneumophila, M. catarrhalis, P.mirabilis, P. aeruginosa, C. pneumoniae, M. pneumoniae, A. anitratus, A.baumannii, A. calcoaceticus, A. lwoffii, B. pertussis, C. diversus, C.freundii, E. aerogenes, E. agglomerans, K. oxytoca, M. morganii, P.vulgaris, P. rettaeri, P. stuartii, P. fluorescens among others. It isenvisioned that the MDT inhibitors identified in the present inventionmay be used in combination with levofloxacin to inhibit or reduce thegrowvth of some or all of these organisms.

In particular, levofloxacin has been indicated for the treatment olindividuals with mild, moderate and severe infection caused by strainsof the designated microorganisms. Particular indications include acutemaxillary sinusitis. acute bacterial exacerbation of bronchitis,acquired pneumonia, uncomplicated skin and skin structure infections,complicated urinary tract infection, and acute pyelonephritis.

The usual dose of Levaquin™ is 500 mg administration by slow infusionover a 60 minute period every 24 hours or as determined by the physicianaccording to the appropriate creatinine clearance. Levaquin™ tablets maybe given as 500 mg orally every 24 hours. The skilled artisan isreferred to the Physicians Desk Reference, (52nd edition 1998,incorporated herein by reference) for more details on amounts andduration of doses.

Norfloxacin is sold under the clinical name Chibroxin™ (Merck & Co.) asan opthalmic solution and as Noroxin™ (Merck & Co.) tablets for oraladministration. In fasting healthy volunteers at least 30-40% of theoral dose of Noroxin is absorbed. Absorption is rapid following singledoses of 200 mg 400 mg and 800 mg. At the respective single doses, themean peak serum and plasma concentrations of 0.8, 1.5 and 2.4 μg/ml areattained approximately one hour after dosing.

Norfloxacin has been shown to be active against most strains ofStaphylococcus aureus, Staphylococcus epidermidis, Staphylococcuswarnerii and Streptococcus pneumoniae Gram positive bacteria.Gram-negative bacteria against which Norfloxacin is clinically usefulinclude Acinetobacter calcoaceticus, Aeromonas hydrophila, Haemophilusinfluenza, Proteus mirabilis, Pseudomonas aeruginos and Serratia.Norfloxacin also has been shown to be valuable in vitro against Bacilluscereus, Entercoccus faecalis (formerly Streptococcus faecalis),Staphylococcus saprophyticus (all Gram-positive bacteria) andCitrobacter diversus, Citrobacter freundii, Edwardsiella tarda,Enterobacter aerogenes, Enterobacter cloacac, Escherichia coli, Hafniaalvei, Haemophilus aegyptius (Koch-Weeks bacillus), Klebsiella oxytoca,Klebsiella pneumoniae, Klebsiella rhinoscleromatis, Morganella morganii,Neisseria gonorrhoeae, Proteus vulgaris, Providencia alcalifaciens,Providencica rettgeri, Providencia stuartii, Salmonella typhi, Vibriocholerae, Vibrio parahemolyticus, Yersinia enterocolitica (Gram negativebacteria).

Norfloxacin is indicated for the treatment of adults with urinary tractinfections, sexually transmitted disease and prostatitis. For moredetailed disclosure on the specific microorganisms mediating theseinfections the skilled artisan is referred to the Physicians DeskReference, (pp. 607-608; 52nd edition, 1998, incorporated herein byreference). Norfloxacin tablets may be administered in a single dose ormultiple doses. the recommended dose is a 400 mg tablet once daily forbetween about 1 day to about 28 days depending on the nature of theinfection. The skilled artisan is referred to the Physicians DeskReference, (52nd edition, 1998, incorporated herein by reference) formore details on amounts and duration of doses.

Ciprofloxacin is available in opthalmic solution, intravenous injectionsolution and tablet formulation. Cipro™ is a broad spectrumfluoroquinolone that is available in 100 mg, 250 mg, 500 mg, and 750 mgcoated tablets which are rapidly and well absorbed from thegastrointestinal tract after oral administration.

Ciprofloxacin has been shown to be active against most strains of thefollowing microorganisms both in vilro and in clinical infections.Aerobic gram-positive bacteria against which ciprofloxacin is activeinclude Enteococcus faecalis, Staphylococcus epidermidis, Staphylococcussaprophyticus, Streptococcus pneumoniae, Streptococcus pyogenes,Staphylococcus aureus. Ciprofloxacin is clinically bacteriotoxic againstvarious aerobic gram-negative bacteria including Campylobacter jejuni,Citrobacter diversus, Citrobacter freundii, Enterobacter cloacae,Esherichia coli, Haemophilus influezae, Haemophilus parainfluenzae,Klebsiella pneumoniae, Moraxella catarrhalis, Morganella morganii,Nisserica gonorrhoeae, Proteus mirabilis, Proteus vulgaris, Proivdenciarettgeri, Providencia stuartii, Pseudomonas aeruginosa, Salmonellatyphi, Serratica marcensens Shigella boydii, Shigella dysenteriae,Shigella flexneri and Shigella sonnei. Ciprofloxacin exhibits in vitrominimal inhibitory concentrations (MICs) of ≦1 μg/mL against most (≧90%)strains of the following microorganisms; however, the safety andeffectiveness of ciprofloxacin in treating clinical infections due tothese microorganisms have not been established in adequate andwell-controlled clinical trials. These bacteria include AcinetobacterIwoffi, Aeromonacs caviae, Aeromronas caviae, Aeromonas hydrophila,Brucella melitensis, Campylobacter coli, Edwardsilla tarda, Haemophilusducreyi, Klebsiella oxytoca, Legionella penumophila, Neisseriameningitidis, Neisseria meningitidis, Pasteurella multocida, Salmonellcaenteritidis, Vibrio cholerae, Vibrio paraphaemolyticus, Vibriovulnificus, Yersinia enterocolitica.

Cipro™ is indicated for the treatment of infection such as acutesinusitis, lower respiratory infections, urinary tract infection, acuteuncomplicated cystitis in females, chronic bacterial prostatitis,complicated intra-abdominal infections, skin and skin structureinfections, bone and bone joint infections, infectious diarrhea, typhoidfever and uncomplicated cervical and urethral gonorrhea. For moredetailed disclosure on the specific microorganisms mediating theseinfections the skilled artisan is referred to the Physicians DeskReference, (pp. 607-608; 52nd edition, 1998, incorporated herein byreference).

The dose of Cipro™ for acute sinusitis is 500 mg every 12 hours. Lowerrespiratory infections may be treated with 500 mg every 12 hours. Formore severe or complicated infections, a dose of 750 mg may be givenevery 12 hours. Urinary tract infection may be treated with 250 mg to500 mg every 12 hours depending on the severity of the infection. Acuteuncomplicated cystitis in females usually requires 100 mg every 12hours. This infection 3 days of treatment may be appropriate whereas 7to 14 days is recommended for other urinary tract infections. Treatmentof chronic bacterial prostatitis uses a regimen of 500 mg every 12hours.

The adult dose for complicated intra-abdominal infections is asequential oral therapy in which 500 mg are administered daily. Theskilled artisan is referred to the Physicians Desk Reference. (pp. 608;52nd edition, 1998, incorporated herein by reference) for detailedprotocols of such sequential therapy. Skin and skin structureinfections, infectious diarrhea, typhoid fever and bone and bone jointinfections are generally treated with a daily 500 mg dose, whereasurethral and cervical gonococcal infections may be treated with a single250 mg dose. The Physicians Desk Reference provides a more detailedprotocol of doses, administration time indications and contraindicationsof this and the other fluoroquinolone described.

Floxin™ is the tradename for the intravenous formulation of otloxacin.It is another broad spectrum, widely prescribed fluoroquinolone. Oraland intravenous administration appears to be similar and comparable inextent of exposure to that observed when an equal (mg/mg) dose isadministered. Thus, the oral and i.v. routes of administration can beconsidered interchangeable.

Floxin™ has been shown to be effective against the following bacteria S.aureus, S. pneumoniae, S. pyogenes, as wells as C. diversus, E.aerogenes, E. coli, H. influenzae, K. pneumonia, N, gonorrhoeae, P.mirabilis and P. Auruginosa. Floxin I.V also has been show to be usefulagainst Staphylococaus epidermidis, Staphylococcus haemolyticus,Staphyloccus saprophyticus, Acinetobacter calcoaceticus, Aeromonascaviae, Aeromonas hydrophila, Bordetella parapertussis, Bordetellapertussis, Citrobacter freundii, Enterobacter cloacae, Haemophilusducreyi, Klebsiella oxytoca, Moraxella catarrhalis, Morganella morganii,Proteus vulgaris, Providencia rettgeri, Providencia stuartii, SerratiaMarcescens and Vibrio parhaemolyticus.

Floxin™ is indicated in acute bacterial exacerbation of chronicbronchitis, community acquired pneumonia, uncomplicated skin and skinstructure infections, acute and uncomplicated urethral and cervicalgonorrhea, nongonoccocal urethritis and cervicitis, mixed infections ofthe urethra and cervix, acute pelvic inflammatory disease, uncomplicatedcystitis, complicated urinary tract infections, and prostatitis. Formore detailed disclosure on the specific microorganisms mediating theseinfections the skilled artisan is referred to the Physicians DeskReference, (pp. 1990-1991; 52nd edition, 1998, incorporated herein byreference)

According to the manufacturers instructions (Ortho-McNeil), Floxin™ I.V.should only be administered by intravenous infusion and may not be usedfor intramuscular, intrathecal, intra peritoneal or subcutaneousadministration. The Floxin™ injection should be administered slowly overa period of not less than 60 minutes. The usual doses of Floxin™ is 200mg to 400 mg administered by slow infusion every 12 hours tor patientspresenting mild to moderate infections and normal renal function. Thusthe dosage of Floxin may vary from 400 mg to 600 mg per day and theduration of treatment can be from between 1 day to as much as 6 weeks.For specific details regarding the dosages and duration ofadministration for particular indications the skilled artisan isreferred to page 1993 of the Physicians Desk Reference. (52nd edition,1998, incorporated herein by reference). Oral formulations of Floxin®also are available and the skilled artisan is referred to page 1997 ofthe Physicians Desk Reference for additional disclosure regardingdosages and duration of administration.

Penetrex™ is yet another commercially available fluoroquinolone with abroad spectrum specificity. Penetrex™ is the tradename for enoxacin andis available in an oral formulation. Enoxacin is an inhibitor of thebacterial enzyme DNA gyrase and is a bactericidal agent. Enoxacin may beactive against pathogens resistant to drugs that act by differentmechanisms.

Penetrex™ has been shown to be active against most strains of thefollowing organisms both in vitro and in clinical infections inStaphylococcus epidermidis and Staphylococcus saprophyticus(Gram-positive aerobes) and Enterobacter cloacae, Escherichia coli,Klebsiella pneumoniae, Neisseria gonorrhoeae, Proteus mirabilis,Pseudomnonas aeruginosa (Gram-negative aerobes). In addition, enoxacinexhibits in vitro minimum inhibitory concentrations (MICs) of 2.0 μg/mLor less against most strains of certain other organisms; however, thesafety and effectiveness of enoxacin in treating clinical infections dueto these organisms have not been established in adequate and wellcontrolled trials, these Gram negative aerobes include Aeromonashydrophila, Citrobacter diversus, Citrobacter freundii, Citrobacterkoseri, Enterobacter aerogenes, Haemophilus ducreyi, Klebsiella oxyloca,Klebsiella ozaenae, Morganella morganii, Proteus vulgaris, Providenciastuartii, Providencia alcalifaciens, Serratia marcescens, Serratiaproteomaculans (formerly S. Liquefaciens)

Penetrex™ is indicated for the treatment of adults presenting sexuallytransmitted diseases and urinary tract infections. Specific dosingdetails may be obtained from the Physicians Desk Reference p2379-2380.

Lomefloxacin is available as Mexaquin® in tablet form for oraladministration. Mexaquin is available as a film coated tablet containing400 mg lomefloxacin base. Lomefloxacin is a bactericidal agent with invitro activity against a wide range of Gram-negative and Gram-positiveorganisms. The bactericidal action of lomefloxacin results frominterference with the activity of the bacterial enzyme DNA gyrase, whichis needed for the transcription and replication of bacterial DNA. Theminimum bactericidal concentration (MBC) generally does not exceed theminimum inhibitory concentration (MIC) by more than a factor of 2,except for staphylococci, which usually have MBCs 2 to 4 times the MIC.

Lomefloxacin has been shown to be active against most strains of thefollowing organisms both in vitro and in clinical infections:Staphylococcus saprophyticus Gram positive bacteria and a longer list ofGram negative bacteria including Citrobacter diversus, Enterobactercloacae, Escherichia Klebsiella, pneumoniae coli, Haemophilusinfluenzae, Moraxella (Branhamella) catarrhalis, Proteus mirabilis,Pseudomonas aeruginosa (urinary tract only). Lomefloxacin exhibits invitro MICs of 2 μg/ml or less against most strains of the followingorganisms; however, the safety and effectiveness of lomefloxacin intreating clinical infections due to these organisms have not beenestablished in adequate and well-controlled trials. In vitro data isavailable against Staphylococcus aureus (includingmethicillin-resistanit strains) Staphylococcus epidermidis (includingmethicillin-resistant strains) Gram positive aerobes and various Gramnegative bacteria including Aeromonas hydrophila, Citrobacter freundii,Enterobacter aerogenes, Enterobracter agglomerans, Haemophiltusparainfluenzae, Hafnia alvei, Klebsiella oxytoca, Klebsiella ozaenae,Morganella morganni, Serratia liquefaciens, Proteus vulgaris,Providencia alcalifaciens, Providencia rettgeri and Serratiamarcesescens.

Maxaquin™ tablets are indicated for the treatment of adults with mild tomoderate infections caused by susceptible strains of microorganisms inconditions such as lower respiratory tract infections and urinary tractinfections. Maxaquin™ has been particularly indicated in the preventionand prophylaxis of transrectal prostate biopsy and transurethralsurgical procedures. On both instances a 400 mg single dose may beorally administered between 1 to 6 hours prior to the operativeprocedure. For additional details on administration protocols theskilled artisan is referred to the Physicians Desk Reference pp.2744-2748.

The fluoroquinolones discussed above are exemplary fluoroquinolones thatmay be used in combination with the MDT inhibitors of the presentinvention. It is understood that the MDT inhibitory compositions of thepresent invention may be used in combination with any fluoroquinolone inorder to potentiate the effect of that fluoroquinolone and/or circumventor prevent resistance to such a drug. Further, it is contemplated thatany of the infections listed herein above or any other infection that istreatable by fluoroquinolone administration will be amenable totreatment with the MDT inhibitors of the present invention incombination with fluoroquinolone treatment.

6. Combination Therapy

Bacterial intrinsic and acquired resistance to antibiotics represents amajor problem in the clinical management of bacterial infections. Thisresistance is mediated at least in part due to the proficiency of themultidrug efflux proteins that are now known to be abundant in bacteria.There are numerous antibiotic ajents that would be excellent therapeuticagents in combating bacterial infection but for the their active effluxfrom the bacterial cells by the action of these MDT proteins. Thus, oneof the goals of current chemotherapeutic research is to find ways ofimproving the efficacy of existing bactericidal compounds againstbacterial infection.

One way of achieving such a beneficial therapeutic outcome is to combinetraditional antibiotics with agents that inhibit the efflux activity ofthe multidrug transporter. Such combination antibiotic therapy would beconceptually similar to the already widely used combinations of β-lactamor cephalosporin antibiotics with inhibitors of β-lactamase. In fact.one such combination, augmentin, has become the most frequentlyprescribed antibiotic preparation in the United States. Moreparticularly, it is a goal of the present invention to improve theefficacy of fluoroquinolone activity. The inventors propose that theclinical use of fluoroquinolones in combination with an inhibitor ofmultidrug transporters should dramatically improve the clinical efficacyof these antibiotics by both reducing their effective concentrationseveral fold (shifting it well below their practically achievable tissuelevels) and preventing the emergence of drug-resistant variants. Morespecifically the present invention provides combinations of afluoroquinolone and an inhibitor of multidrug transporter(MDT inhibitor)for combating Gram positive infection. Equally in mycologicalapplications, the MDT inhibitors may be combined with other anti-fungaltreatments.

To kill bacterial cells, inhibit bacterial cell growth, or otherwisereverse or reduce the suppressing effect on the emergence ofdrug-resistant variants bacterial species using, the methods andcompositions of the present invention. one would generally contact a“target” cell with an MDT inhibitor and at least one fluoroquinolone.The antifungal applications of the present invention will be similarexcept that the cells being killed, inhibited or suppressed will befungal cells. The compositions would be provided in a combined amounteffective to kill or inhibit bacterial cell growth. This process mayinvolve contacting the cells with the MDT inhibitor and thefluoroquinolone(s) or other bactericidal factor(s) at the same time.This may be achieved by contacting the cell with a single composition orpharmacological formulation that includes both agents, or by contactingthe cell with two distinct compositions or formulations. at the sametime, wherein one composition includes the MDT inhibitor and the otherincludes the fluoroquinolone.

The MDT inhibitor treatment may precede or follow the otherfluoroquinolone by intervals ranging from minutes to hours to days. Inembodiments where the fluoroquinolone and MDT inhibitor are administeredseparately, one would generally ensure that a significant period of timedid not expire between the time of each delivery, such that thefluoroquinolone and MDT inhibitor would still be able to exert anadvantageously combined effect on abrogating the bacterial infection. InSuch instances, it is contemplated that one would administer bothmodalities within about 12-24 hours of each other and, more preferably,within about 6-12 hours of each other, with a delay time of only about12 hours being most preferred, It may be that in order to sensitize thebacterial cells to the fluoroquinolone treatment. the MDT inhibitol isadministered for a sufficient period of time (1, 2 3, 4, 5, 6, 7, 8, 12,24 hours) prior to the fluoroquinolone treatment. In some situations, itmay be desirable to extend the time period for treatment significantly,however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1,2,3, 4, 5, 6, 7 or 8) lapse between the respective administrations.Equally it may be necessary to administer multiple doses of the MDTinhibitor in order to sensitize the bacterial cells to thefluoroquinolone treatment.

It also is conceivable that more than one administration of either MDTinhibitor or the fluoroquinolone will be desired. Various combinationsmay be employed, where the MDT inhibitor is “A” and the fluoroquinoloneis “B”, as exemplified below:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B

A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A

A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B

Other combinations are contemplated. Again, to achieve bacterial cellkilling, both agents are delivered to a cell in a combined amounteffective to kill the cell and remove the infection.

Agents or factors suitable for use in a combined therapy are anyfluoroquinolone chemical compound or treatment method that inducesdamage when applied to a bacterial cell. More particularly, the presentinvention uses fluoroquiniolone in combination with the MDT inhibitorsof the present invention. Such fluoroquinoloncs include but are notlimited to pefloxacin, norfloxacin, ciprofloxacin, ofloxacin,levofloxacin, enoxacin, fleroxacin, lomefloxacin, temofloxacin,amifloxacin, tosufloxacin, flumequine, rufloxacin. clinafloxacin and thelike.

In certain embodiments, the MDT inhibitors of the present invention maybe used in combination with antifungal agents to combat fungalinfection. Such antifungal agents include but are not limited toamphotericin B. flucytosine, ketoconazole, miconazole, itraconazole,fluconazole, griseofluconiazole, clotrimazole, econazole, terconazole.butaconazole, nystatin, haloprogin, loprox, natamycin, undecylenic acidand others.

In treating a bacterial infection according to the invention, one wouldcontact the bacterial cells with a fluoroquinolone agent in addition tothe MDT inhibitor. This may be achieved by contacting the bacterialcells with the agent by administering to the subject a therapeuticallyeffective amount of a pharmaceutical composition comprising afluoroquinolone compound and a therapeutically effective amount of theMDT inhibitor. Similarly, in treating a fungal infection according tothe invention, one would contact the fungal cells with an antimycoticagent in addition to the MDT inhibitor. The antifungal or antibacterialagent may be prepared and used as a combined therapeutic composition. orkit, by combining it with a MDT inhibitor, as described above.

The skilled artisan is directed to “the Physicians Desk Reference” 52ndEdition, in order to find detailed specific disclosure regardingparticular fluoroquinolonles. Some variation in dosage will necessarilyoccur depending on the condition of the subject beiln treated. Theperson responsible for administration will, in any event, determine theappropriate dose for the individual subject. Moreover, for humanadministration. preparations should meet sterility, pyrogenicity,general safety and purity standards as required by the FDA Office ofBiologics standards.

The inventors propose that the regional delivery of MDT inhibitor and/orthe fluoroquinolone compositions to patients with Gram positivebacterial infection will be a very efficient method for delivering atherapeutically effective composition to counteract the clinicaldisease. Alternatively, systemic delivery of MDT inhibitor and/or thefluoroquinolone may be the most appropriate method of achievingtherapeutic benefit from the compositions of the present invention.Likewise, the MDT inhibitor and/or antimycotic agent compositions may beadministered to patient with fungal infection as a regional delivery,systemic delivery or topical application.

It also should be pointed out that any of the foregoing MDT inhibitorsmay prove useful by themselves in treating a bacterial or fungalinfection. In this regard, reference to chemotherapeutics in combinationalso should be read as a contemplation that these approaches may beemployed separately.

7. Pharmaceutical Administration

Pharmaceutical compositions of the present invention will generallycomprise an effective amount of the MDT inhibitor dissolved or dispersedin a pharmaceutically acceptable carrier or aqueous medium. Thepharmaceutical composition may further comprise a fluoroquinolonecomposition.

The phrases “pharmaceutically or pharmacologically acceptable” refer tomolecular entities and compositions that do not produce an adverse,allergic or other untoward reaction when administered to an animal, or ahuman, as appropriate. As used herein, “pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents and the like. The use of such media and agents for pharmaceuticalactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active ingredient,its use in the therapeutic compositions is contemplated. Supplementaryactive ingredients also can be incorporated into the compositions.

The MDT inhibitor of thlc present invention will often be formulated forparenteral administration, e.g., formulated for injection via theintravenous, intramuscular. sub-cutaneous or other such routes,including direct instillation into an infected or diseased site. Thepreparation of an aqueous composition that contains an MDT inhibitoragent as an active ingredient will be known to those of skill in the artin light of the present disclosure. Typically, such compositions can beprepared as injectables, either as liquid solutions or suspensions;solid forms suitable for using to prepare solutions or suspensions uponthe addition of a liquid prior to injection also can be prepared; andthe preparations also can be emulsified.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant. such as hydroxypropylcellulose. Dispersions also can beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi.

The MDT inhibitor compositions can be formulated into a composition in aneutral or salt form. Pharmaceutically acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups alsocan be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylarnine. trimethylamine, histidine, procaine and thelike.

The carrier also can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. Formulations are easily administered in a variety of dosageforms, such as the type of injectable solutions described above, butdrug release capsules and the like also can be employed.

Suitable pharmaceutical compositions in accordance with the inventionwill generally include an amount of the MDT inhibitor admixed with anacceptable pharmaceutical diluenit or excipient, such as a sterileaqueous solution, to give a range of final concentrations, depending onthe intended use. The techniques of preparation are generally well knownin the art as exemplified by Remington's Pharmaceutical Sciences. 16thEd. Mack Publishing Company, 1980, incorporated herein by reference. Itshould be appreciated that, for human administration, preparationsshould meet sterility, pyrogenicity, general safety and purity standardsas required by the FDA Office of Biological Standards.

The therapeutically effective doses are readily determinable using ananimal model, as shown in the studies detailed herein. Experimentalanimals bearing bacterial or fungal infection are frequently used tooptimize appropriate therapeutic doses prior to translating to aclinical environment. Such models are known to be very reliable inpredicting effective anti-bacterial and anti-flungal strategies.

In addition to the compounds formulated for parenteral administration,such as intravenous or intramuscular injection, other pharmaceuticallyacceptable forms also are contemplated, e.g. tablets or other solids fororal administration, time release capsules, liposomal forms and thelike. Other pharmaceutical formulations may also be used, dependent onthe condition to be treated.

For oral administration the MDT inhibitors of the present invention maybe incorporated with excipients and used in the form of non-ingestiblemouthwashes and dentifrices. A mouthwash may be prepared incorporatingthe active ingredient in the required amount in an appropriate solvent,such as a sodium borate solution (Dobell's Solution). Alternatively, theactive ingredient may be incorporated into an antiseptic wash containingsodium borate, glycerin and potassium bicarbonate. The active ingredientmay also be dispersed in dentifrices, including: gels, pastes, powdersand slurries. The active ingredient may be added in a therapeuticallyeffective amount to a paste dentifrice that may include water, binders,abrasives, flavoring agents, foaming agents, and humectants.

The present invention also provides therapeutic kits comprising the MDTinhibitors described herein. Such kits will generally contain, insuitable container means. a pharmaceutically acceptable formulation ofat least one MDT inhibitor in accordance with the invention. The kitsmay also contain other pharmaceutically acceptable formulations, such asthose containing antibiotics such as fluoroquinolones; and any one ormore of a range of chemotherapeutic drugs.

The kits may have a single container means that contains the MDTinhibitor, with or without any additional components, or they may havedistinct container means for each desired agent. Certain preferred kitsof the present invention include a MDT inhibitor, packaged in a kit foruse in combination with the co-administration of fluoroquinolones. Insuch kits, the MDT inhibitor and the fluoroquinolone may bepre-complexed, either in a molar equivalent combination, or with onecomponent in excess of the other; or each of the MDT inhibitor andfluoroquinolone components of the kit may be maintained separatelywithin distinct containers prior to administration to a patient. Otherpreferred kits include any MDT inhibitor of the present invention incombination with a “classic” chemotherapeutic agent. This is exemplaryof the considerations that are applicable to the preparation of all suchMDT inhibitor kits and kit combinations in general.

When the components of the kit are provided in one or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly preferred. However, the componentsof the kit may be provided as dried powder(s). When reagents orcomponents are provided as a dry powder, the powder can be reconstitutedby the addition of a suitable solvent. It is envisioned that the solventmay also be provided in another container means.

The container means of the kit will generally include at least one vial,test tube, flask, bottle, syringe or other container means, into whichthe MDT inhibitor, and any other desired agent, may be placed and,preferably, suitably aliquoted. Where additional components areincluded, the kit will also generally contain a second vial or othercontainer into which these are placed, enabling the administration ofseparated designed doses. The kits may also comprise a second/thirdcontainer means for containing a sterile, pharmaceutically acceptablebuffer or other diluent.

The kits may also contain a means by which to administer the MDTinhibitor to an animal or patient, e.g., one or more needles orsyringes, or even an eye dropper, pipette, or other such like apparatus,from which the formulation may be injected into the animal or applied toa diseased area of the body. The kits of the present invention will alsotypically include a means for containing the vials, or such like, andother component, in close confinement for commercial sale, such as,e.g., injection or blow-molded plastic containers into which the desiredvials and other apparatus are placed and retained.

Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without eparting from the spirit and scope of theinvention.

Example 1 CoMFA (3D-QSAR) Analysis for Potential Inhibitors of theMultidrug Transporter NorA

The DiverSet™ library of chemical compounds was screened for compoundseffective, at concentrations of 20 μg/ml or less, in reversing theresistance of a specially created B. subtilis strain NA to the NorAsubstrate ethidium bromide. 399 compounds were suggested as potentialinhibitors. Of these 399, 54 showed activity at 5 μg/mL or less, whilethe others showed moderate to little activity at 10-20 μg/mL. Three ofthe most potent compounds are shown below as INF55 with an indolemoiety; a urea compound, INF271; and INF240, possessing an aromaticamide functional group. A large number of other compounds in this setcould be classified according to these three, and since it is unclearwhether NorA has more than one potential binding site, the compoundswere subdivided into the indoles (and nitroindoles), the ureas, and thearomatic amides. These three classes of compounds were evaluated usingthe activity data provided and the CoMFA fields generated to see if a3D-QSAR relationship was present.

Computational Methods

All of the compounds in the study were initially optimized using MM3,and a conformational search done to find the lowest energy conformer.Subsequently, this lowest energy conformer was then reoptimized (also atMM3), and used as the inventors' initial geometry. The partial atomiccharges were generated using AM1 (Dewar et al., 1985) within Sybyl 6.4(Tripos, Inc., St. Louis, Mo.). The CoMFA analysis was performed usingSybyl 6.4. One of the big concerns for a CoMFA analysis is the choice ofsuperposition. The inventors chose to use DISCO (Martin et al., 1993) tooverlay the molecules, which finds common pharmacaphore points withinthe set of compounds (and their conformers). More than one DISCO modelwas generally found, and the model with the best fit (overlay) was usedfor each class of compounds. This overlay of molecules was then used togenerate the CoMFA steric and electrostatic fields. Using the CoMFA andactivity data, a PLS analysis was performed to check the validity of themodel. First cross-validation PLS was run to optimize the number ofcomponents and to check the r² _(cv). For the inventors' analysis the r²_(cv) value was no lower than 0.42 for any of the inventors' systems.The r² and F value were then determined with the optimal number ofcomponents and with no cross-validation. These values are presented inTable 1 for all of the systems.

Results

Indoles. The nitroindoles and indoles were the first class of compoundsanalyzed. Initially these two were studied separately. There were only asmall number of nitroindoles studied. 13 compounds, resulting in amediocre model (see Table 1). There were a total of 40 indoles that didnot possess a substituent at the nitrogen position that also weremodeled resulting in a better model than the nitroindoles. However, thebest model (from PLS analysis, Table 1) generated was upon the inclusionof the nitroindoles with the indoles, where the inventors had a total of49 compounds. This model was then used to evaluate the steric andelectrostatic fields for all and used for predicting activities of otherindoles. The steric and electrostatic field contour maps generated byCoMFA are shown with INF55 in FIG. 1 and FIG. 2.

TABLE 1 Results from PLS analysis for the Systems Evaluated Number ofStandard Compounds error of Probe System in analysis R² F value estimateatom Nitroindoles 13 0.781 39.181 0.273 sp³C(+1) Indoles 40 0.953371.603 0.059 sp³C(+1) Nitroindoles + 49 0.995 1611.971 0.034 sp³C(+1)Indoles Ureas 28 0.951 195.320 0.085 sp³C(+1) 28 0.993 846.187 0.028O(−1) Biphenyl 132 0.913 265.566 0.106 sp³C(+1) Ureas Aromatic 50 0.960366.416 0.064 O(−1) Amides + ureas

TABLE 2 Favorable and Unfavorable Positions for Substituents on IndolesSubstituent Favored position(s) Unfavorable position(s) Model 1: —Cl(Phat R₁₎ R₅ —CH₃(Ph at R₁₎ R₂ R₅ 2-naphthyl R₁ anisole R₁(ortho) R₁(para)t-butyl R₁ Model 2: —Cl R₁ —CH₃ R₁ R₂ —OMe R₂ R₁ -Ph Fused at R₁-R₂Model 3: (Ph at R₁) R₄ -X(F or Cl), —SO₂Me, —CO₂H, —CX₃ (Ph at R₁) R₄—SO₃H, —NH₂ (2-naphthyl at R₁) R₁ —CX₃

Overall, using the models above, enhanced activity is suggested whenmore bulky groups are placed at R₁, and where the nitro group is onModels 1 and 2, or in the vicillity of R₃, R₄, and R₅ for the moregeneral Model 3. It appears that it would be best if R₂ and R₆ weresimply hydrogens. Electrostatically, the substituent(s) at the R₃, R₄,or R₅ position is optimal if it were a strong electron withdrawing groupso as to result in a more negative region in these areas and a morepositive region surrounding the indole ring center. There doesn't seemto be much preference for charge on the substituent at R₁, except for afew, very small areas where a negative charge may be favored.

From these results, 30 new indole compounds were analyzed using thismodel to predict their activity to see (1) if there is a bettersubstituent at R₁ besides a phenyl group. and (2) if there is a morefavorable substituent for the R₄ position in place of a nitro group.Table 2 summarizes the groups that had an extreme impact on increasingor decreasing the activity. In terms of R₁, 2-naphthyl and o-anisole,XII and VIII, were predicted to show comparable activity to INF55. Twoothers were predicted to be slightly less potent than INF55, having anaphthyl group fused to the indolc rings, and the addition of a methylgroup at the R. position along with the phenyl at the R₁, IX and XI.Therefore, the best group at R₁ would either be a phenyl or the2-naphthyl. Using either the phenyl group or the 2-naphthyl at R₁, R₄was then varied to find a more suitable group in place of the nitro. Thesulfonyl group, shown in IV, is predicted to be the best of those lookedat, not quite as active as the nitro group but it would still show atleast a two-fold improvement over reserpine, known to be active at 5μg/mL. Also of mention is placing an amine at the R₄ position, XIIIwhich is predicted to be almost as active as the sulfonyl group. Othercompounds that were predicted to be comparable to the activity ofreserpine were the trifluoro- and trichloromethyl groups at R₄, and inthese cases the 2-naphthyl group at R₁ as in XIV, showed better activitythan the phenyl group.

Overall for the indoles the CoMFA model suggests placing a moreelectronegative group at the R₄ position and an aromatic ring system atR₁. The aromatic ring system at R₁ would increase the size of themolecule, but at the same time keep the molecule relatively rigid andflat, an apparently favored 3D structure.

Ureas. Using original activity data along with activity data from newlysynthesized biphenyl ureas, a new CoMFA model was generated. The bestmodel for the urea system came from 132 compounds, using an sp³ carbonatom with a charge of +1, see Table 1. The steric field contour map withINF271, 276 placed within the field is shown if FIG. 3. Observation ofthe steric fields associated with the latest CoMFA model shows thefollowing:

This new model reinforces the original one in that the bulkiersubstituents need to be placed away from the urea center. In addition itappears that the carbonyl group needs to remain “sterically unhindered”,i.e. no substituenits at R₅ or R₆. The electrostatic field contour mapis shown in FIG. 4. This updated electrostatic field suggests thefollowing:

The electrostatic indications are that negatively charged groups, i.e.electron-withdrawing groups are favored in the region surrounding R₇,R₈, and R₉. Positively charged groups, those possessing very littleelectronegativity or being electron-donating groups are favored in theR₁ and R₃ positions. Positively charged groups are also favored in theR₆ and R₇ region as long as they are distanced fiom the aromatic ring.From the above results and based on the substituents known to enhancethe activity of the biphellyl ureas, the following model is suggested.

Suggested Model for Ureas:

In this model, two positions are crucial, R₁ needs to have an alkoxygroup and there needs to be an aromatic group at R₈ such as phelnyl,benzoyl, or a fused aromatic ring at R₇-R₈. There is also evidence thatsubstituents can also be placed at R₃ and R₉. Currently the inventorsplan to analyze yet more biphenyl ureas to further optimize thesesubstituents for these positions.

Aromatic amides. Due to having the same pharmacaphore points withinDISCO as the ureas, new CoMFA model and PLS analysis was performed usingboth aromatic amides and ureas. A total of 50 compounds were analyzed.The CoMFA results of the steric and electrostatic fields are in FIG. 5and FIG. 6, placing INF240 within these contours. Using the model below,CoMFA suggests the following: bulky groups limited to R₂, and a longchain R₆ where the bulkier substituents are away from the aromatic amideitself. Also, if R₆ is not a long chain (less than 4 carbons) thensubstitution on the aromatic ring also can take place at the R₄position. The R₄/R₆ combination must be fairly rigid and planar so as tokeep the bulky groups in one region. Electrostatics once again indicatesa large positive and large negative region on either side of the amidegroup(as in the urea system), and doesn't provide any indications awayfrom this group.

Summary

The CoMFA analysis has given suggestions for the indole systems, whichhave fairly rigid structures. From the original test-set, the sets ofstructurally diverse ureas and aromatic amide CoMFA's did not generatevery specific models. However, upon further analysis, a large number ofbiphenyl ureas were found to have increased activity. From our compoundset, 142 biphenyl ureas were found to be active (non-toxic and active atless than 10μg/ml). Of these 142 biphenyl ureas, 132 of them generated areliable CoMFA analysis. The most recent CoMFA study for the ureasprovides information regarding more optimal substituenits for enhancingthe activity. The aromatic amides do not seem to be as promising of alead model as either the indoles of ureas, most likely due to the muchhigher degree of flexibility of the compounds. However, limiting thetype of aromatic amide, in much the same way as suggested for the ureas,may also be one alternative to better describing this system.

Example 2 Characterization of the Inhibitory Action of IdentifiedCompounds

The present Example provides instructions regarding the measurement ofthe inhibitory activity of identified compounds.

Quantitation of Effects of Combination of Five Selected Inhibitors withEthidium on Bacterial Growth.

Synergy testing was performed with the strain NA by checkerboardtitration in microtiter plates using two-fold serial broth microdilution(Eliopoulus and Moellering Jr., 1996). Each candidate inhibitor wastested at 11 concentrations (ranging from 50 ng/ml -50 μg/ml) andethidium was tested at 11 concentrations ranging from 30 ng/ml to 40μg/ml (the MIC for the strain NA). Wells were assessed visually forgrowth after an 18 h incubation period. The Fractional InhibitoryConcentration (FIC) was calculated for each inhibitor and ethidiumcombination. The following formulae were used to calculate the FICindex:${{FIC}\quad {of}\quad {drug}\quad A} = \frac{{MIC}\quad {of}\quad {drug}\quad A\quad {in}\quad {combination}}{{MIC}\quad {of}\quad {drug}\quad A\quad {alone}}$${{FIC}\quad {of}\quad {drug}\quad B} = \frac{{MIC}\quad {of}\quad {drug}\quad B\quad {in}\quad {combination}}{{MIC}\quad {of}\quad {drug}\quad B\quad {alone}}$FIC  index = FIC  of  drug  A + FIC  of  drug  B.

Synergy was defined as an FIC index of <0.5. FIG. 7 shows arepresentative synergy curve obtained for the combination of one of theinhibitors with ethidium. The calculated FIC indices are shown in Table3.

TABLE 3 Synergy results for the combination of NorA inhibitors withethidium. Inhibitor FIC index INF 55 0.08 INF 240 0.16 INF 271 0.07 INF277 0.09 INF 392 0.14

The FIC indices for all five of the candidate inhibitors were <<0.5indicating that these compounds were strongly synergistic in promotingthe bacteriostatic effects of ethidium, which is what would be expectedfor an inhibitor of the ethiditim-resistanice mechanism, NorA in thisparticular case.

Evaluation of Candidate Inhibitors Ability to Promote Toxicity bySuppressing Efflux by NorA.

NA cells over expressing NorA were loaded with ethidium bromide in thepresence of reserpine (20 μg/ml). After washings cells were placed in afluorimeter cuvette with fresh medium. Since ethidium fluoresces onlywhen it is located inside the cell and bound to DNA, cells exhibited arapid decrease in fluorescence due to NorA-mediated ethidium efflux. Asshown in FIG. 8, this decrease in fluorescence was dramaticallyinhibited when cells were allowed to efflux in the presence of reserpineor each of the five tested compounds. The inventors conclude that thelead compounds synergistically promoted the toxicity of ethiditimbromide by inhibiting the efflux of the drug by the NorA multidrugefflux transporter.

Evaluation of Synergism Between Inhibitors and Ciprofloxacin inPromoting Bacteriotoxicity

The inventors have quantitated the effects of the combination of theNorA inhibitors with ciprofloxacin, currently the most widely usedfluoroquiniolone and the second most prescribed antibiotic. Synergytesting was performed by checkerboard titration as described above forethidium. Eleven concentrations of ciprofloxacin ranging from 4 ng/ml to4 μg/ml (two times the MIC) were used with both the NA strain and the S.aureus strain SA1199B which overexpresses NorA from a single chromosomalcopy of the NorA gene (Kaatz et al., 1990). All five compounds acted ina synergistic manner with ciprofloxacin, having FIC indices <0.5 (seeTable 4). TIhe inventors concluded that. similarly to their effects onthe bacteriotoxicity of ethidium, each of the tested NorA inhibitorspromotes the bacteriotoxicity of ciprofloxacin in a synergistic manner.

TABLE 4 Synergy results for the combination of NorA inhibitors withciprofloxacin. Inhibitor INF 55 INF 240 INF 271 INF 277 INF 392 FICindex NA 0.25 0.12 0.12 0.15 0.28 FIC index SA1199B 0.25 0.28 0.18 0.280.15

Evaluate the Effect of the Inhibitors on the Intrinsic Susceptibility ofWild Type S. aureus to Ciprofloxacin.

The expression of NorA in wild type S. aureus confers significantintrinsic resistance to a number of fluoioquinolones includingnorfloxacin and ciprofloxacin (Yamada et al., 1997). The inventorstherefore evaluated whether the newly identified inhibitors couldpotentiate the bacteriotoxic effects of fluoroquinolones in wild type S.aureus. As shown in FIG. 9 all of the identified NorA inhibitorsdecreased the IC₅₀ of ciprofloxacin by at least 3 fold. Thus theclinical use of any of the identified inhibitors in combination withciprofloxacin would likely shift the MIC₉₀ of this antibiotic for S.aureus to well below the clinically achievable concentration. Since thefrequency of emergence of low levet fluoroquinolone resistance candecrease by two orders of magnitude when selecting for a two-folddifference in the MIC (four times versus two times) (Wakabayashi andMitsuhashi, 1994), the inventors next evaluated whether NorA inhibitorscould, by promoting the intracellular accumulation of the antibiotic,decrease the rate of emergence of ciprofloxacin resistant variants.

Effect of Inhibitors on the Rate of Emergence of CiprofloxacinResistance in S. aureus.

The effect of the inhibitors on the rate of emergence of in vitroselected single-step ciprofloxacin resistant mutants of wild type S.aureus SA1199 (Kaatz et al., 1990) was determined. Spontaneous mutantswere obtained 48 h after plating 2-4 33 10¹⁰ SA1199 cells on LB agarplates containing ciprofloxacin at a concentration of 1 μg/ml (2×MIC).TFhe frequency of mutant selection was determined to be 2×10⁻⁹ bycomparing the number of colonies that grew on plates containing the drugwith the number of colonies obtained upon plating appropriate dilutionsin the absence of drug. Similar to the inventors' previous studies withnorfiloxacin (Markham and Neyfakh, 1996), reserpine dramaticallyinhibited the emergence of ciprofloxacin resistance by more than oneorder of magnitude. Importantly, as shown in Table 5, each of the testedinhibitors also decreased the frequency of spontaneous emergence ofciprofloxacin resistance by 50-fold or more. This dramatic effect couldnot be attributed to a toxic effect of the inhibitor since the sameconcentration of inhibitor affected neither the colony forming ability,nor the colony size of SA1199 cells plated in the absence ofciprofloxacin.

TABLE 5 Frequency of emergence of in vitro selected variants of SA1199resistant to two fold the MIC of ciprofloxacin in the absence orpresence of NorA inhibitors. Inhibitor, concentration SpontaneousMutagenized None 2.5 × 10⁻⁹ 2.5 × 10⁻⁸ Reserpine, 20 μg/ml <5 × 10⁻¹¹<2.5 × 10⁻¹⁰ INF 55, 5 μg/ml <5 × 10⁻¹¹ <2.5 × 10⁻¹⁰ INF 240, 5 μg/ml <5× 10⁻¹¹ <2.5 × 10⁻¹⁰ INF 271, 5 μg/ml <5 × 10⁻¹¹ <2.5 × 10⁻¹⁰ INF 277, 5μg/ml <5 × 10⁻¹¹ <2.5 × 10⁻¹⁰ INF 392, 5 μg/ml <5 × 10⁻¹¹ 1 × 10⁻⁹

Ciprofloxacin resistance in first step in vitro selected mutants of S.aureus is predominantly due to specific point mutations in the targetsof this drug, topoisomerase IV and gyrase (Cambau and Gutman, 1993,Ferrero et al., 1994). This explains why chemical mutagenesis of S.aureus by ethylmethane sulfonate increases the rate of emergence ofciprofloxacin-resistant variants by an order of magnitude (Table 5).However, even with mutagenized cells, the NorA inhibitors stronglysuppressed the appearance of drug-resistant colonies. In conclusion, theidentified lead inhibitors, like reserpine, inhibited the emergence offluoroquinolone resistance in S. aureus.

Evaluate the Possibility of NorA Becoming Insensitive to the Inhibitorsby Mutation.

One potential limitation to the combination of an antibiotic with aninhibitor of the resistance mechanism is the possibility of theresistance mechanism developing mutations making it insensitive to theinhibitor. Such a situation has been observed for bacteria which,through mutations in the β-lactamase gene, have developed resistance toAugmentin (a combination of ampicillin and clavulanic acid. an inhibitorof β-lactamase). Similarly, mutations in NorA could theoretically causeNorA to develop resistance to the inhibitor. Indeed, previous studieswith Bmr, a close homolog of NorA, have shown that this multidrug effluxtransporter can mutate to resist the inhibitory effects of reserpinewhile retaining drug eftiux activity (Ahmed et al., 1993).

To evaluate the possibility of such mutations arising in the NorAtransporter the inventors determined the frequency of emergence ofmutants of NorA-overexpressing S. aureus, SA1199B, that retainedresistance to a NorA substrate in spite of the presence of a NorAinhibitor. These cells were chemically multageniized withethiylmetlhanie sulfonate (Markham and Neyfakh, 1996) and 2-4×10⁹ cellswere then selected on plates containing the NorA substrate ethidiumbromide (10 μg/ml—a quarter of the MIC) and either reserpine (20 μg/ml)or one or the five lead inhibitors (5 μg/ml). After a 48 h incubationperiod the number of colonies on each plate was determined.

Mutants insensitive to reserpine arose at a frequency of approximately2×10⁻⁸. As shown in Table 6, mutants insensitive to INF 392 arose at aneven higher frequency. However, very few mutants (2.5-5×10⁻⁹) could beobtained with INF 277 and INF 240, and, most encouragingly, no mutantscould be obtained which were insensitive to either INF 55 or INF 271.This strongly indicates the feasibility of developing an inhibitor towhich NorA would be unable to adapt.

TABLE 6 Summary of the properties of the lead NorA inhibitors InhibitorINF 55 INF 240 INF 271 INF 277 INF 392 Reserpine Effectivity IC₅₀* S.Aureus, μg/ml 1.5 0.8 1.5 0.8 0.1 6.25 Toxicity HeLa IC₅₀, μg/ml 40 2 185 45 N.D Selectivity Index (Toxicity HeLa/Effectivity) 27 2.5 12 6 450N.D. Fold inhibition of emergence of resistance >50 >50 >50 >50 25 >50Frequency of adaptation of NorA <5 × 10⁻¹⁰ <5 × 10⁻⁹ <5 × 10⁻¹⁰ <5 ×10⁻⁹ <5 × 10⁻⁷ <5 × 10⁻⁸ IC₅₀ S. Pneumoniae, μg/ml 5 N.A. 2.5 5 2.5 5*IC₅₀ for S. aureus (SA1199B) and S. pneumoniae (SPC2A) is defined hereas the minimal concentration of drug required to decrease the MIC forciprofloxacin by two fold. N.A. = not active at 5 μg/ml. N.D. = notdetermined.

Example 3 Toxicity Testing of Identified Inhibitors

Here the inven tors proposed to test the toxicity of the identified leadcompounds on several human cell lines. To date, the inventors havedetemined the IC₅₀ of the lead compounds for the HeLa cell line, theresults of which are presented in Table 6. The toxicity of the compoundsat concentrations ranging from 0.7 μ/ml to 100 μg/ml was determined in a96 well plate by adding the compounds to cells 24 h after seeding at adensity of 10⁴ per well. After incubation with the compounds for 3 daysthe effect on cell growth was determined using, the Cell Titer 96AQ_(ueous) MTS-based assay (Promega, Madison, Wis.).

Example 4 Pharmaceutical Compositions of the Present Invention

The MDT composition s of the present invention may be formulated astablets or as solutions for injection as discussed in the pharmaceuticalcompositions section therein above. Trhe present section is intended toprovide illustrative examples of MDT compositions for use in treating asubject with a bacterial or fungal infection. In treating bacterialinfections, these MDT compositions may thus be provided to the subjectin combination with a fluoroqlinolone. The fluoroquinolone may beprovided in a separate composition, or where the biological chemistryallows, the fluoroquinolone may form part of the active ingredients ofthe MDT composition. In treating fungal infection, the MDT compositionsmay be provided to the subject in combination with an antiamycoticagent.

Compositions containing a dose of 100, 200, 300, 400 or 500 mg of an MDTinhibitor of the present invention are prepared as follows. Theappropriately hydrated or dehydrated form of the MDT inhibitor forms theactive ingredient of the composition. In the tablet formulation, anexemplary excipient core may comprise wheat starch, gelatin, talc,magnesium stearate sodium carboxymethylstarch, for a core and thecoating comprising hydroxypropyl methylcellulose, ethyl cellulose,dibutyl sebacate, titanium oxide, talc, polyethylene glycol 600.

In addition to a composition comprising a single MDT inhibitor, it iscontemplated that the active ingredients of the composition may beformulated to include two or more of the MDT inhibitors to provide abroader spectrum of activity.

Furthermore, it is contemplated that the therapeutic compositions of thepresent invention may comprise as an additional active ingredient, oneor more fluoroquilnolone such as for example, pefloxacin, norfioxacin,ciprofloxacin, ofloxacin, sparfloxacin, grepafloxacin, Bay 12-8039,trovafloxacin, DU6859a, sarafloxacin, LB20304. lcvofloxacin, enoxacin,fleroxacin, lomefloxacin, temofloxacini, arnifloxacin, tosufloxacin,flumequinie, rufloxacin, clinafloxacin and the like.

In antimycotic applications, the therapeutic compositions of the presentinvention may comprise as an additional active ingredient, one or moreanti-fungal agent such as amphotericin B., flucytosine, ketoconazole,miconazole, itraconazole, fluconazole, griseofluconazole, nystatin,haloprogin, loprox, natamycin, undecylenic acid and the like.

It is understood that the above formulations are provided by way of anexample. one of skill in the art would be able to formulate acomposition in which the inhibitors identified herein may be placed intoany pharmaceutical carrier for the purposes of therapeutic delivery.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically it will be apparentthat certain agents that are both chemically and physiologically relatedmay be substituted for the agents described herein while the same orsimilar results would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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What is claimed is:
 1. A method for enhancing the antibacterial actionof fluorquinolones comprising contacting a bacterium with an indoleinhibitor of NorA, said indole having the formula:

wherein R₁ is phenyl, 2-napthyl, o-anisole, R₂ is H or CH₃, R₁ and R₂are two napthyl groups fused to the indole ring, R₃ is H, R₄ is NO₂,SO₃H, NH₂, CF₃ or CCl₃, R₅ is H, and R₆ is H.
 2. The method of claim 1,wherein R₁ is phenyl and R₄ is SO₃H or NO₂.
 3. The method of claim 1,wherein R₁ is 2-naphthyl and R₄ is CCl₃ or CF₃.
 4. The method of claim1, wherein R₁ is o-anisole and R₄ is NO₂.
 5. The method of claim 1,wherein R₁ and R₂ are two naphthyl groups fused to the indole ring. 6.The method of claim 1, wherein R₁ is phenyl and R₂ is CH₃.
 7. The methodof claim 1, wherein said bacterium is Streptococcus pneumonia,Enterococcus faecalis, Staphylococcus aureus, Streptococcus pygenes,Escherichia coli, Psuedomonas aeruginosa, Staphylococcus epidermis,Mycobacterium tuberculosis, Mycobacterium smegmatis and Serratiamarcesens.
 8. An indole having the formula:

wherein R₁ is phenyl, ₂-napthyl, o-anisole, R₂ is H or CH₃, R₁ and R₂are two napthyl groups fused to the indole ring, R₃ is H, R₄ is NO₂,SO₃H, NH₂, CF₃ or CCl₃, R₅ is H, and R₆ is H.
 9. The indole of claim 8,wherein R₁ is phenyl and R₄ is SO₃H or NO₂.
 10. The indole of claim 8,wherein R₁ is 2-naphthyl and R₄ is CCl₃ or CF₃.
 11. The indole of claim8, wherein R₁ is o-anisole and R₄ is NO₂.
 12. The indole of claim 8,wherein R₁ and R₂ are two naphthyl groups fused to the indole ring. 13.The indole of claim 8, wherein R₁ is phenyl and R₂ is CH₃.
 14. Apharmaceutical composition comprising a fluoroquinolone and an indoleinhibitor of NorA, said indole having the formula:

wherein R₁ is phenyl, 2-napthyl, o-anisole, R₂ is H or CH₃, R₁ and R₂are two napthyl groups fused to the indole ring, R₃ is H, R₄ is NO₂,SO₃H, NH₂, CF₃ or CCl₃, R₅ is H, and R₆ is H.